Culture reagent composition and culture method for blood system malignant tumor organoid

Abstract

The invention belongs to the technical field of biological medicines, and particularly relates to a culture reagent composition and a culture method for a malignant tumor organ of a blood system. The invention firstly provides a reagent composition for culturing a hematological malignant tumor organoid, wherein the hematological malignant tumor comprises acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition comprises a bone marrow microenvironment simulation matrix reagent and a specific culture medium. By adopting the reagent composition provided by the invention, the hematoma organs can be efficiently cultured, and the survival rate is high. And compared with Matrigel matrigel commonly used in the field, the bone marrow microenvironment simulation matrix component adopted by the invention enables phenotypic differentiation of hematoma organs to be more accurate.

Description

[0001] Divisional application The present application is a divisional application of the Chinese patent application for invention with the application number 2025107269727, the application date of June 3, 2025, and the invention name of "Culture method and application of blood system malignant tumor organoids". TECHNICAL FIELD

[0002] The present application belongs to the technical field of biological medicine, and specifically relates to a culture reagent composition and culture method of blood system malignant tumor organoids. BACKGROUND

[0003] Blood system malignant tumor refers to the malignant tumor occurring in the blood system (bone marrow, hematopoietic tissue and lymphoid tissue), mainly including leukemia, lymphoma and multiple myeloma, etc. Among them, leukemia and lymphoma are both ranked in the top ten of global tumor morbidity and mortality. At present, blood system malignant tumor is classified according to morphology, immunophenotype, cytogenetics and molecular abnormalities, and clinical characteristics, mainly divided into myeloid or lymphoid and acute or chronic. Leukemia and lymphoma have many similarities. When tumor cells (lymphoblasts) involve blood and bone marrow (defined as more than 20% of bone marrow blast cells), the disease can manifest as leukemia; when the original cells mainly infiltrate the extramedullary tissue to form tissue masses, the disease can manifest as lymphoma.

[0004] Patient-derived cell culture 3D models have been reported in a variety of solid tumors, but blood system malignant tumor 3D models (such as leukemia, lymphoma) are rarely reported. Although there are documents reporting the use of iPSC-derived or healthy donor hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) to construct bone marrow organoids to simulate the normal bone marrow microenvironment. However, the construction of blood system malignant tumor organoids is more difficult. The construction of blood system malignant tumor organoids often needs to overcome key challenges such as spontaneous differentiation / apoptosis of malignant cells, clonal selection deviation and insufficient microenvironment simulation; in addition, blood system malignant tumor is easy to die or heterodifferentiate during the culture process, and heterodifferentiation will lead to deviation of drug sensitivity test results and clinical response, and thus lead to inaccurate drug test results based on organoid models.

[0005] In summary, it is urgent to propose new methods and strategies to improve the above-mentioned deficiencies of the prior art. SUMMARY

[0006] The purpose of the present application is to provide a culture reagent composition and culture method of blood system malignant tumor organoids, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The present application specifically adopts the following technical solutions.

[0007] The first aspect of the present application provides a composition for culturing a blood malignant tumor organoid.

[0008] A reagent composition for culturing a blood malignant tumor organoid, the blood malignant tumor including acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition including a bone marrow microenvironment mimetic matrix reagent and a specific medium; The bone marrow microenvironment mimetic matrix reagent has a function of providing an environment for correct differentiation of an immunophenotype of the cultured blood malignant tumor organoid; The bone marrow microenvironment mimetic matrix reagent is a gelatin methacryloyl (GelMA) solution containing a photoinitiator; The specific medium is composed of a basic medium and a cytokine; the basic medium is a serum-free medium for hematopoietic stem cells; the cytokine includes a first cytokine composition and a specific cytokine; the first cytokine composition includes penicillin / streptomycin, stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4; the specific cytokine is interleukin 7 or thrombopoietin; in the first cytokine composition, the concentration ratio of the stem cell factor to the FMS-like tyrosine kinase 3 is 1:1; the concentration ratio of the interleukin 3, the fibroblast growth factor 2, and the bone morphogenetic protein 4 is 1:1:1; The bone marrow microenvironment mimetic matrix reagent and the specific medium are independently provided, and when used for culturing the blood malignant tumor organoid, the tumor cells are first added to the bone marrow microenvironment mimetic matrix reagent for photocuring, and then the specific medium is added; When the tumor cells are acute lymphoblastic leukemia cells, the specific cytokine is interleukin 7; When the tumor cells are acute myeloid leukemia cells, the specific cytokine is thrombopoietin.

[0009] Further, the mass concentration of the gelatin methacryloyl solution is 5%-10%; the mass concentration of the photoinitiator is 0.01%-0.05%.

[0010] As a preferred, the photoinitiator is a photoinitiator capable of initiating ultraviolet photocuring; specifically, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, but is not limited.

[0011] As a preferred, the mass concentration of the gelatin methacryloyl solution is 5%; the mass concentration of the photoinitiator is 0.05%.

[0012] Further, the first cytokine composition specifically consists of the following components: 1%-3% penicillin / streptomycin, 20-30 ng / mL stem cell factor, 20-30 ng / mL FMS-like tyrosine kinase 3, 10-15 ng / mL interleukin 3, 10-15 ng / mL fibroblast growth factor 2, and 10-15 ng / mL bone morphogenetic protein 4; and the specific cytokine is 10-15 ng / mL interleukin 7 or 20-30 ng / mL thrombopoietin.

[0013] As a preferred, the first cytokine composition specifically consists of the following components: 1% penicillin / streptomycin, 25 ng / mL stem cell factor, 25 ng / mL FMS-like tyrosine kinase 3, 10 ng / mL interleukin 3, 10 ng / mL fibroblast growth factor 2, and 10 ng / mL bone morphogenetic protein 4; and the specific cytokine is 10 ng / mL interleukin 7 or 25 ng / mL thrombopoietin.

[0014] Another aspect of the present application is to provide a method for culturing a blood malignant tumor organoid.

[0015] The method for culturing a blood malignant tumor organoid with an immune phenotype close to that of a patient based on the above-mentioned reagent composition for culturing a blood malignant tumor organoid comprises the following steps: S01: taking a tumor cell sample, which includes an acute lymphoblastic leukemia cell sample or an acute myeloid leukemia cell sample, resuspending in a bone marrow microenvironment simulation matrix reagent of the reagent composition, and ultraviolet curing; 30000-50000 tumor cells per 25 μL of the bone marrow microenvironment simulation matrix reagent; S02: adding a specific culture medium to the bone marrow microenvironment simulation matrix reagent containing the tumor cell sample for blood malignant tumor organoid culture; S03: identifying the immune phenotype of the cultured blood malignant tumor organoid; and screening a blood malignant tumor organoid with an immune phenotype close to that of the patient's bone marrow cells.

[0016] Further, the tumor cell sample is a tumor cell sample derived from bone marrow or a tumor cell sample derived from peripheral blood.

[0017] Further, the immune phenotype identification includes co-incubating the cultured blood malignant tumor organoid with a blood malignant tumor specific antibody, detecting the expression of the specific antibody on the blood malignant tumor organoid; and taking the expression of the specific antibody on the patient's bone marrow cells as a comparison. The expression amount of the specific antibody on the blood malignant tumor organoid is not more than 10% different compared with the patient's bone marrow cells.

[0018] Further, the specific antibody includes CD34.

[0019] The blood malignant tumor organoid obtained by the above method is applied to the preparation of a preclinical drug resistance detection model.

[0020] Beneficial technical effects: The present application first provides a culture reagent composition for culturing blood system malignant tumor organoids (mainly acute lymphoblastic leukemia or acute myeloid leukemia), which contains innovative culture medium components and bone marrow microenvironment simulation matrix components, wherein the culture medium uses a first cytokine composition (specifically stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2 and bone morphogenetic protein 4) with a simplified composition but a high concentration, and a specific cytokine with a high concentration is added, so that the blood tumor organoid can be efficiently cultured with a high survival rate. Further, the innovative bone marrow microenvironment simulation matrix component makes the phenotype differentiation of the blood tumor organoid more accurate compared with the Matrigel matrix gel commonly used in the art.

[0021] Secondly, based on the above-mentioned culture reagent composition, the present application also provides a method for standardizing the construction of blood system malignant tumor organoids. According to the method provided by the present application, a plurality of AML\ALL organoids have been cultured, which proves that the method of the present application has good repeatability. The blood tumor organoid cultured by the present application is non-epithelial-derived bone marrow cells, and achieves rapid growth in 7 days of culture, which has an advantage over the current culture of solid tumor organoids in the art which mainly uses epithelial-derived cells and has a longer culture period. In addition, there are few reports on the successful culture of blood tumor organoids at present, so the technical solution of the present application has significant novelty.

[0022] Finally, the present application also performs phenotype identification on the cultured blood tumor organoid, and the screened blood tumor organoid is similar to the patient's cell immunophenotype, which can be used for preparing a drug resistance detection model. The drug resistance detection model can more accurately screen which drug is more sensitive to the patient with drug resistance in clinic, thereby increasing the effect of disease treatment. And through experiments, it has been verified that the blood tumor organoid prepared by the present application can more accurately reflect the drug resistance, which is closely related to the reagent containing in the composition provided by the present application, which promotes the correct differentiation of the immunophenotype of the blood tumor organoid. BRIEF DESCRIPTION OF DRAWINGS

[0023] In order to more clearly illustrate the technical solutions in the embodiments of the present application or the prior art, the following will briefly introduce the drawings needed to be used in the embodiments or prior art description. In all the drawings, similar elements or parts are generally identified by similar reference signs. In the drawings, each element or part is not necessarily drawn according to the actual proportion. Obviously, the drawings described below are some embodiments of the present application, and for those skilled in the art, other drawings can also be obtained from these drawings without creative labor.

[0024] Figure 1 Bright field image of growth of acute lymphoblastic leukemia organoids in one of the embodiments of the present application (scale bar 50 μm); Figure 2 Results of sensitivity experiments of ALL organoids cultured in one of the embodiments of the present application to different anti-tumor drugs; Figure 3 Bright field image of growth of acute myeloid leukemia in different culture conditions in one of the embodiments of the present application; Figure 4 Results of drug sensitivity experiments of different AML organoids to venetoclax in one of the embodiments of the present application; Figure 5 Results of sensitivity experiments of AML organoids in different culture systems to drugs in one of the embodiments of the present application; Figure 6 Results of sensitivity experiments of AML organoids cultured in one of the embodiments of the present application to different anti-tumor drugs. DETAILED DESCRIPTION

[0025] In order to make the objects, technical solutions and advantages of the embodiments of the present application clearer, the following will combine the drawings in the embodiments of the present application to clearly and completely describe the technical solutions in the embodiments of the present application. Obviously, the described embodiments are some of the embodiments of the present application, rather than all the embodiments. Based on the embodiments in the present application, all other embodiments obtained by those skilled in the art without creative labor fall within the scope of protection of the present application.

[0026] In this document, "and / or" includes any and all combinations of one or more of the associated items.

[0027] In this document, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0028] As used in this specification, the term "about", when used in reference to a particular value, typically means + / - 5% of the stated value, more typically + / - 4% of the stated value, more typically + / - 3% of the stated value, more typically + / - 2% of the stated value, even more typically + / - 1% of the stated value, and even more typically + / - 0.5% of the stated value.

[0029] In this specification, certain embodiments can be disclosed in one format in terms of a range. It is to be understood that such a "range in terms of" description is used for convenience and brevity and should not be construed as a rigid limitation on the scope of the disclosure. Accordingly, the description of a range should be considered as having specifically disclosed all possible subranges as well as individual numerical values within that range. For example, a description of a range 1-6 should be considered to have specifically disclosed the subranges like from 1 to 3, from 1 to 4, from 1 to 5, from 2 to 4, from 2 to 6, from 3 to 6 etc., as well as the individual numbers 1, 2, 3, 4, 5, and 6. This applies regardless of the breadth of the range.

[0030] Glossary: The "close to the immune phenotype of the patient" in the present application refers to the result of flow cytometry identification showing similarity to the immune phenotype (surface marker) of the tumor cells of the patient.

[0031] Example 1 This example provides a method for culturing, identifying and drug screening of blood system malignant tumor organoids.

[0032] 1. A method for culturing blood tumor organoids.

[0033] S01: Take the bone marrow or peripheral blood sample of the patient, obtain the tumor cell sample after density gradient centrifugation; resuspend the tumor cell sample in an appropriate amount of red blood cell lysis solution, lyse on ice for 3 min, add DPBS to terminate lysis. Precool the centrifuge at 600 g, 4 ℃ for 5 min, remove the supernatant. Resuspend the cells in an appropriate amount of bone marrow microenvironment simulation matrix, which is a 5% GelMA (containing 0.05% photoinitiator LAP) solution, seed in a 48-well plate, and solidify.

[0034] S02: Add 200 μL of SFEM II medium (StemSpan SFEM II serum-free medium for hematopoietic stem cells) and cytokines for blood tumor organoid culture; the cytokines include 1% P / S (penicillin / streptomycin), 25 ng / mL SCF (stem cell factor), 25 ng / mL FLT3 (FMS-like tyrosine kinase 3), 10 ng / mL IL-3 (interleukin 3), 10 ng / mL FGF2 (fibroblast growth factor 2), 10 ng / mL BMP4 (bone morphogenetic protein 4), 10 ng / mL IL-7 (interleukin 7), or 25 ng / mL TPO (thrombopoietin). Replace the organoid culture medium every 2-3 days, and subculture the obtained primary organoids after 7-10 days of culture. The obtained organoids are blood system malignant tumor organoids, referred to as blood tumor organoids.

[0035] 2. Identification of blood tumor organoids.

[0036] Flow cytometry is used to analyze the cell composition of the organoids and detect the expression of antibodies CD45, CD34, etc.

[0037] 3. Drug screening.

[0038] The blood tumor organoids successfully cultured above are digested with a digestive solution and washed with DPBS buffer, then centrifuged at 600 g and 4°C for 5 min to remove the supernatant. The collected cells are counted, resuspended in culture medium, and seeded in a 96-well plate at 1000 organoids per well, and the drug to be tested is added. After 5 days, cell viability test is performed.

[0039] Example 2 This example provides an example of constructing an acute lymphoblastic leukemia (ALL) organoid.

[0040] 1. Primary culture: (1) The patient signs the informed consent form, and the patient's bone marrow sample is taken and placed at room temperature for a period of time, then washed with a room temperature PBS dilution.

[0041] (2) Add 15 mL of sample density gradient separation solution to a 50 mL centrifuge tube, then slowly add the diluted blood to the separation solution surface.

[0042] (3) Centrifuge at 400 g for 30 minutes at room temperature.

[0043] (4) Take the centrifuge tube out of the centrifuge, at this time the centrifuge tube liquid surface is divided into four layers, from top to bottom, the first layer is plasma layer, the second layer is mononuclear cell layer, the third layer is separation liquid, and the fourth layer is red blood cell layer, discard the uppermost layer, and transfer the mononuclear cell layer to another centrifuge tube, add one volume of PBS, and centrifuge at 300 g for 15 minutes at room temperature.

[0044] (5) After centrifugation, take the bottom precipitate, add 2-3 mL of red blood cell lysis solution, lyse on ice for 3-5 minutes, and centrifuge at 300 g for 15 minutes.

[0045] (6) After cell counting, take a proper amount of cells, resuspend them in 5% GelMA (containing 0.05% light initiator LAP), and inoculate them in a 48-well plate (25 μL of 5% GelMA resuspended 50,000 cells), irradiate with ultraviolet light for 60 s for solidification, add ALL organoid-specific medium for culture, and replace the medium every 2-3 days. Passage every 7-10 days. The results of 7-day culture are shown in Figure 1 , which shows that the ALL organoids grow rapidly in 7 days.

[0046] ALL organoid-specific medium: SFEM II medium (StemSpan SFEM II hematopoietic stem cell serum-free medium) and specific cytokines; the specific cytokines are composed of 1% P / S (penicillin / streptomycin), 25 ng / mL SCF (stem cell factor), 25 ng / mL FLT3 (FMS-like tyrosine kinase 3), 10 ng / mL IL-3 (interleukin 3), 10 ng / mL FGF2 (fibroblast growth factor 2), 10 ng / mL BMP4 (bone morphogenetic protein 4), and 10 ng / mL IL-7 (interleukin 7).

[0047] As a control, after cell counting, take a proper amount of cells, resuspend them in Matrigel, and inoculate them in a 48-well plate (25 μL of Matrigel resuspended 50,000 cells), and after solidification in the incubator, add the ALL organoid-specific medium for culture, and replace the medium every 2-3 days. Passage every 7-10 days.

[0048] 2. Subculture: (1) After the organoids grow to a proper amount, perform digestion and expansion, aspirate the medium in the cell culture plate, use a 1 mL suction head to scrape the organoid-GelMA mixed glue droplet, collect it in a 15 mL centrifuge tube, blow and mix the suspension, centrifuge at 300 g for 5 min.

[0049] (2) Discard the supernatant, add 2-3 mL of GelMA digestion solution (EFL) according to the volume of GelMA after centrifugation, and gently blow 4-5 times with a gun head.

[0050] (3) Put into 37°C water bath to digest, observe under microscope until digestion is complete, then terminate digestion, centrifuge at 300g for 5 min.

[0051] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, centrifuge at 300g for 5 min.

[0052] (5) According to the number of cell clusters, resuspend the cells with 5% GelMA, inoculate in a 48-well plate, irradiate with ultraviolet light for 60 s to solidify, add ALL organoid-specific medium, replace the liquid every 2-3 days. Passage every 7-10 days.

[0053] 3. Cryopreservation: (1) When the organoids grow to a proper number, remove the medium in the cell culture plate, take PBS, use a 1 mL suction head to scrape the organoid-GelMA mixed glue droplet, collect in a 15 mL centrifuge tube, mix the suspension by blowing with a suction head, centrifuge at 300g for 5 min.

[0054] (2) Discard the supernatant and observe the amount of GelMA. According to the volume of GelMA after centrifugation, add 2-3 mL of organoid digestion solution, and gently blow 4-5 times with a gun head that has been rinsed with rinse solution.

[0055] (3) Put into 37°C water bath to digest, observe under microscope until digestion is complete, then terminate digestion, centrifuge at 300g for 5 min.

[0056] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, centrifuge at 300g for 5 min.

[0057] (5) Cryopreserve in liquid nitrogen.

[0058] 4. Thawing: (1) Take the cryopreservation tube out of liquid nitrogen, thaw in a 37°C water bath.

[0059] (2) After thawing is complete, transfer the organoid cryopreservation solution containing cells to a 50 mL centrifuge tube, slowly add 9 mL of pre-cooled PBS, centrifuge at 600g for 5 min.

[0060] (3) Discard the supernatant, transfer the cell pellet to a 15 mL centrifuge tube with PBS, centrifuge at 600g for 5 min.

[0061] (4) Discard the supernatant, according to the number of cell clusters, resuspend the cells with 5% GelMA, inoculate in a 48-well plate, irradiate with ultraviolet light for 30 s to solidify, add ALL organoid-specific medium, replace the liquid every 2-3 days. Passage every 7-10 days.

[0062] 5. Identification: Flow cytometry: Take the same patient's bone marrow sample and the ALL organoids cultured in the above steps to digest into single cells, and incubate them with selected antibodies (CD34, HLA-DR, CD33, cD3, CD7, CD13) to specifically bind the antibodies to the antigens on the surface of the cells. Turn on the flow cytometer and preheat and calibrate the instrument; place the labeled sample in the flow cytometer for detection; see Table 1 for analysis and identification results.

[0063] Diagnosis of ALL standard: The proportion of blast cells (compared to nucleated cells) in bone marrow ≥ 20% is diagnosed as ALL, which in this embodiment refers to the marker CD34 of blast cells. It should be noted that ALL can be divided into different subtypes, but this embodiment does not identify the typing of ALL, only whether the cultured organoids are ALL.

[0064] Table 1 Analysis and identification of ALL organoid immunophenotype The results show that the binding of ALL organoids cultured in 5% GelMA to antibodies is almost identical to that of ALL patient bone marrow cells, indicating that the phenotype of the cultured ALL organoids is very close to that of the real patient. Conversely, the binding of ALL organoids cultured in Matrigel matrix gel to antibodies is quite different from that of ALL patient bone marrow cells, indicating that the phenotype of the ALL organoids cultured by this method is quite different from that of the real patient.

[0065] 6. Drug screening: (1) When the above ALL organoids grow to sufficient amount, collect the organoids, digest them into single cells, and count them.

[0066] (2) After centrifugation, discard the supernatant, and according to the counting results, ensure that the plating density is 500-1000 organoids per well.

[0067] (3) Prepare an appropriate amount of ALL organoid-specific culture medium in advance.

[0068] (4) Use a 1000 μL pipette to mix the cell suspension with the adjusted density, and add it to the 96-well plate at a volume of 100 μL / well, trying to ensure that the number of organoids in each well is uniform.

[0069] (5) The blank group only adds 100 μL of medium without cells per well.

[0070] (6) After plating, PBS can be added to the surrounding wells to maintain humidity; place the 96-well plate in the incubator and wait for drug addition.

[0071] (7) Using a 200 μL pipette, add the prepared drug solution of each target concentration venetoclax: 10, 20, 40, 100 (unit μM); vinblastine sulfate: 0.001, 0.01, 0.1, 1, 10 (unit nM) into the corresponding drug groups in turn, 100 μL of drug solution per well, the tip along the side wall of the well, and note that the drug solution should be mixed well by blowing several times before adding.

[0072] (8) After aspirating the drug solution of different concentrations, replace the tip for the next step.

[0073] (9) The negative group and the blank group only add 100 μL of culture medium.

[0074] (10) After adding the drug, gently tap the culture plate to mix the drug solution with the cells for sufficient reaction.

[0075] (11) Place the well plate in the incubator, and perform ATP detection after 5 days of drug action.

[0076] (12) The above cultured ALL organoids are derived from the bone marrow cells of a patient clinically resistant to venetoclax, and the drug detection results of the cultured ALL organoids are shown in Figure 2 , and the results show that the organoid model is more sensitive to vinblastine sulfate than to venetoclax, and shows resistance to venetoclax, which is consistent with the patient's drug response.

[0077] Example 3 This example provides an example of culturing and drug screening of acute myeloid leukemia (AML) organoids.

[0078] 1. Culturing of acute myeloid leukemia (AML) organoids: the method is the same as that of Example 2, and the AML organoid-specific culture medium used is as follows.

[0079] The AML organoid-specific culture medium is SFEM II medium (StemSpan SFEM II serum-free medium for hematopoietic stem cells) and specific cytokines; the specific cytokines are composed of 1% P / S (penicillin / streptomycin), 25 ng / mL SCF (stem cell factor), 25 ng / mL FLT3 (FMS-like tyrosine kinase 3), 10 ng / mL IL-3 (interleukin 3), 10 ng / mL FGF2 (fibroblast growth factor 2), 10 ng / mL BMP4 (bone morphogenetic protein 4), and 25 ng / mL TPO (thrombopoietin).

[0080] AML organoid control medium: SFEM II medium (StemSpan SFEM II serum-free medium for hematopoietic stem cells) and specific cytokines; the specific cytokines are composed of 1% P / S (penicillin / streptomycin), 25 ng / mL SCF (stem cell factor), 25 ng / mL FLT3 (FMS-like tyrosine kinase 3), 10 ng / mL IL-3 (interleukin 3), 10 ng / mL FGF2 (fibroblast growth factor 2), and 10 ng / mL BMP4 (bone morphogenetic protein 4).

[0081] 2. Different culture condition test: AML organoids were cultured in a similar manner to Example 2, wherein a portion of the AML organoids were cultured using the AML organoid control medium; another portion of the AML organoids were cultured using the AML organoid specific medium; images were collected after 3 days of culture. The results are shown in Figure 3 , where it can be seen that the organoids in the TPO group grow faster and are larger in size.

[0082] On the other hand, a portion of the AML organoids were resuspended in Matrigel and added with the AML organoid specific medium and cultured for 3 days; a portion of the AML organoids were resuspended in 5% GelMA and seeded in a 48-well plate for photocuring and added with the AML organoid specific medium and cultured for 3 days. The results are shown in Figure 3 , where it can be seen that the 5% GelMA has larger organoid sphere size and more number of organoid spheres than Matrigel.

[0083] The cultured AML organoids were subjected to immunophenotyping to screen out organoids close to the immunophenotype of bone marrow cells of the same patient for subsequent experiments.

[0084] 3. Digestion of organoids: (1) When the organoids grow to an adequate amount, collect the organoids, digest them to single cells, and count them.

[0085] (2) After centrifugation, discard the supernatant, and according to the counting results, ensure that the plating density is 500-1000 organoids per well.

[0086] 4. Cell plating: (1) Prepare an appropriate amount of AML organoid specific medium in advance.

[0087] (2) Using a 1000 μL pipette, mix the cell suspension with adjusted density by blowing, and add it to the 96-well plate at a volume of 100 μL / well, trying to ensure that the number of organoids in each well is uniform.

[0088] (3) The blank group only adds 100 μL of medium without cells per well.

[0089] (4) After the plate is laid, PBS can be added to the surrounding wells to maintain humidity; place the 96-well plate in an incubator and wait for the drugs to be added. The drugs used in this example are veneclade, azacitidine, decitabine, and giretinib.

[0090] 5. Drug effects: (1) Using a 200μL pipette, add the prepared target concentrations of Veneclair: 50, 5, 0.5, 0.05, 0.005, 0.0005, 0.00005 (unit μM); and Azacitidine, Decitabine, Gefitinib: 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 (unit μM) to the corresponding dosing groups in sequence, 100μL of drug solution per well, and add the drug solution along the side wall of the well with the pipette tip. Note that you should blow the pipette several times before adding the drug solution to mix it evenly.

[0091] (2) After drawing up different concentrations of drug solution, change the nozzle to proceed to the next step.

[0092] (3) Only 100 μL of culture medium was added to the negative group and the blank group.

[0093] (4) After the drug is added, you can gently tap the culture plate to mix the drug solution with the cells for a full reaction.

[0094] (5) Place the well plate in an incubator and perform the test after 5 days of drug treatment.

[0095] 6. Testing: Five days later, an ATP test was performed.

[0096] 7. Data Analysis: Based on the ATP detection results, data analysis was performed using GraphPad Prism, and the experimental results are as follows: Figure 4 As shown, AML organoid 1 was derived from bone marrow cells of patients who had not received drug treatment; AML organoid 2 was derived from bone marrow cells of patients clinically resistant to veneclade. In vitro drug susceptibility testing results showed that the models successfully preserved the drug resistance characteristics of the original tumors. Notably, different organoids exhibited significant heterogeneity to the same drug, while traditional cell line models (MOLM-13 and HL60) showed high homogeneity in drug susceptibility responses, demonstrating that organoids are superior to traditional cell lines in simulating individualized clinical differences.

[0097] Furthermore, to verify the influence of the microenvironment matrix on drug resistance phenotype, the experiment further compared two culture systems: matrix gel and 5% GelMA hydrogel. Figure 5). Experiments show that: 1) in the experimental group adding venetoclax, AML organoids 2 cultured in Matrigel are sensitive to venetoclax, that is, the drug resistance phenotype is reversed, indicating that the phenotype of the organoids is deviated during the culture process (consistent with the results of culturing ALL in Example 2), while AML organoids 2 cultured in 5% GelMA hydrogel are resistant to venetoclax, which is consistent with the drug resistance phenotype of the patient. 2) In the Gilteritinib treatment group, AML organoids 2 cultured in 5% GelMA hydrogel are significantly more sensitive to Gilteritinib, proving that using organoid models close to the patient's phenotype can more accurately screen suitable treatment drugs for drug-resistant patients.

[0098] Further, Figure 6 It is shown that the patient-derived organoid model without drug treatment is used for sensitivity detection of various drugs, and the results show that the organoids have different responses to different drugs, that is, different sensitivities to different antitumor drugs. Since it has been proved in the previous experiments that the blood tumor organoid model cultured by the method of the application has a closer phenotype to the patient, therefore, using such blood tumor organoids for preclinical drug screening tests, the results have high reference value.

[0099] It should be noted that in this document, the terms "comprising", "including", or any other variant thereof are intended to cover non-exclusive inclusion, so that a process, method, article or apparatus including a series of elements includes not only those elements, but also other elements not explicitly listed, or other elements inherent to such a process, method, article or apparatus. Without more limitations, the element defined by the statement "including a…" does not exclude the presence of other identical elements in the process, method, article or apparatus including the element.

[0100] The embodiments of the application are described above in conjunction with the drawings, but the application is not limited to the specific embodiments described above, which are only illustrative and not restrictive, and those of ordinary skill in the art can make many forms under the inspiration of the application without departing from the purpose of the application and the scope protected by the claims, which are all within the protection of the application.

Claims

1. A reagent composition for hematological malignancy organoid culture, characterized by, The blood malignancy includes acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition includes a bone marrow microenvironment simulation matrix reagent and a specific culture medium; The bone marrow microenvironment simulation matrix reagent is a methacrylated gelatin solution containing a photoinitiator; The specific culture medium is composed of a basic culture medium and a cytokine; the basic culture medium is a serum-free culture medium for hematopoietic stem cells; the cytokine includes a first cytokine composition and a specific cytokine; The first cytokine composition includes penicillin / streptomycin, stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4; the specific cytokine is interleukin 7 or thrombopoietin; In the first cytokine composition, the concentration ratio of stem cell factor to FMS-like tyrosine kinase 3 is 1:1; the concentration ratio of interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4 is 1:1:1; The bone marrow microenvironment simulation matrix reagent and the specific culture medium are independently arranged, and when used for blood malignancy organoid culture, the tumor cells are first added to the bone marrow microenvironment simulation matrix reagent for photocuring, and then the specific culture medium is added; When the tumor cells are acute lymphoblastic leukemia cells, the specific cytokine is interleukin 7; When the tumor cells are acute myeloid leukemia cells, the specific cytokine is thrombopoietin.

2. The reagent composition of claim 1, wherein The mass concentration of the methacrylated gelatin solution is 5%-10%; the mass concentration of the photoinitiator is 0.01%-0.05%.

3. The reagent composition of claim 2, wherein The photoinitiator is lithium phenyl(2,4,6-trimethylbenzoyl) phosphate.

4. The reagent composition of claim 2, wherein The mass concentration of the methacrylated gelatin solution is 5%; the mass concentration of the photoinitiator is 0.05%.

5. The reagent composition of claim 1, wherein The first cytokine composition specifically consists of the following components: 1%-3% penicillin / streptomycin, 20-30 ng / mL stem cell factor, 20-30 ng / mL FMS-like tyrosine kinase 3, 10-15 ng / mL interleukin 3, 10-15 ng / mL fibroblast growth factor 2, and 10-15 ng / mL bone morphogenetic protein 4; the specific cytokine is 10-15 ng / mL interleukin 7 or 20-30 ng / mL thrombopoietin.

6. The reagent composition of claim 5, wherein The first cytokine composition specifically consists of the following components: 1% penicillin / streptomycin, 25 ng / mL stem cell factor, 25 ng / mL FMS-like tyrosine kinase 3, 10 ng / mL interleukin 3, 10 ng / mL fibroblast growth factor 2, and 10 ng / mL bone morphogenetic protein 4; the specific cytokine is 10 ng / mL interleukin 7 or 25 ng / mL thrombopoietin.

7. A method of culturing a blood malignancy organoid having a close resemblance to the immune phenotype of a patient based on the reagent composition of any one of claims 1-6, characterized in that, The method comprises the following steps: S01: taking a tumor cell sample, which includes an acute lymphoblastic leukemia cell sample or an acute myeloid leukemia cell sample, resuspending in a bone marrow microenvironment mimetic matrix reagent of the reagent composition, and ultraviolet light curing; resuspending 30000-50000 tumor cell samples per 25 μL of the bone marrow microenvironment mimetic matrix reagent; S02: adding a specific culture medium to the bone marrow microenvironment mimetic matrix reagent containing the tumor cell sample for blood malignant tumor organoid culture; S03: immunophenotypic identification of the blood malignant tumor organoid obtained by culture; screening out a blood malignant tumor organoid close to the immunophenotype of the patient's bone marrow cells.

8. The method of claim 7, wherein, The tumor cell sample is a bone marrow-derived tumor cell sample or a peripheral blood-derived tumor cell sample.

9. The method of claim 7, wherein, The immunophenotypic identification includes co-incubating the blood malignant tumor organoid obtained by culture with a blood malignant tumor-specific antibody, detecting the expression of the specific antibody on the blood malignant tumor organoid; and taking the expression of the specific antibody on the patient's bone marrow cells as a comparison; The expression amount of the specific antibody on the blood malignant tumor organoid differs by no more than 10% compared with the patient's bone marrow cells.

10. The method of claim 9, wherein, The specific antibody includes CD34.

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