Culture method and application of blood system malignant tumor organoid

Abstract

The invention belongs to the technical field of biological medicine, and particularly relates to a culture method and application of a hematological malignant tumor organoid. The culture method comprises the following steps: S01, obtaining a tumor cell sample; s02, preparing a bone marrow microenvironment simulation matrix, and resuspending tumor cells; s03, a specific culture medium is added for hematoma organoid culture; s04, performing phenotype identification on the cultured hematological malignant tumor organoid; and hematological malignant tumor organs with phenotypes close to those of bone marrow cells of the patient are screened out. According to the method system disclosed by the invention, the hematoma organoid is successfully cultured and is further subjected to phenotype identification, and the screened hematoma organoid is close to the cellular immune phenotype of a patient and can be used for accurate detection of clinical patient drug sensitivity and effective test of potential effective target drugs.

Description

Technical Field

[0001] The present invention belongs to the technical field of biomedicine, and particularly relates to a method for culturing and application of hematological system malignant tumor organoids. Background Art

[0002] Hematological system malignant tumors refer to malignant tumors occurring in the hematological system (bone marrow, hematopoietic tissue and lymphoid tissue), mainly including leukemia, lymphoma and multiple myeloma, etc. Among them, leukemia and lymphoma are both ranked among the top ten in terms of global tumor incidence and mortality. At present, hematological system malignant tumors are classified according to morphology, immunophenotype, cytogenetics and molecular abnormalities as well as clinical features, mainly divided into myeloid or lymphoid and acute or chronic. Leukemia and lymphoma have many similarities. When tumor cells (lymphoblasts) involve the blood and bone marrow (defined as more than 20% of bone marrow blasts), the disease can be manifested as leukemia; when the blasts mainly infiltrate extramedullary tissues to form tissue masses, the disease can be manifested as lymphoma.

[0003] The 3D models cultured from patient-derived cells have been reported in a variety of solid tumors, but there are few reports on 3D models of hematological system malignant tumors (such as leukemia, lymphoma). Although there are literatures reporting the construction of bone marrow organoids using iPSC-derived or healthy donor hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) to simulate the normal bone marrow microenvironment. However, the construction of hematological system malignant tumor organoids is more difficult. The construction of hematological system malignant tumor organoids often needs to overcome key challenges such as spontaneous differentiation / apoptosis of malignant cells, clonal selection bias and insufficient bionic degree of the microenvironment. Specifically, hematological system malignant tumor cells are prone to spontaneous differentiation or apoptosis in vitro culture due to detachment from the native microenvironment, resulting in the inability of organoids to truly simulate the molecular characteristics of patient tumors. Secondly, due to the high clonal heterogeneity of hematological malignancies, conventional culture media will cause clonal selection bias, that is, while the dominant clones over-proliferate, rare drug-resistant subpopulations or cancer stem cells are gradually eliminated, reducing the predictive value of the constructed model for the recurrence mechanism. In addition, hematological malignant tumor organoids are very easy to die or undergo heterotypic differentiation during the culture process, and heterotypic differentiation will lead to deviations between the results of drug sensitivity tests and clinical responses, so precise regulation of the culture system is required. On the other hand, different from the construction of bone marrow organoids, the cells for constructing hematological system malignant tumor organoids include bone marrow or peripheral blood cells from patients. During the culture process, it is necessary to specifically maintain the proliferation, heterogeneity and pathological characteristics of malignant clone cells in a three-dimensional system, and at the same time simulate tumor-related stromal cells, extracellular matrix and abnormal cytokine networks, which also increases the construction difficulty.

[0004] In summary, there is an urgent need to propose a method for standardizing the construction of hematological system malignant tumor organoids to supplement the deficiencies of the existing technology. Summary of the Invention

[0005] The object of the present invention is to provide a method for culturing hematological system malignant tumor organoids and their application in preclinical drug screening, to partially solve or alleviate the above deficiencies in the prior art. The present invention specifically adopts the following technical solutions.

[0006] In the first aspect of the present invention, there is provided a method for culturing hematological system malignant tumor organoids.

[0007] A method for culturing hematological malignant tumor organoids with an immunophenotype close to that of the patient, comprising the following steps: S01: Taking a cell sample of a hematological malignant tumor patient, the cell sample including a tumor cell sample derived from bone marrow and / or a tumor cell sample derived from peripheral blood; S02: Preparing a bone marrow microenvironment-mimicking matrix, the bone marrow microenvironment-mimicking matrix being a methacrylated gelatin (GelMA) solution containing a photoinitiator, and resuspending the tumor cells in the bone marrow microenvironment-mimicking matrix solution for ultraviolet light curing; S03: Adding a specific medium to the bone marrow microenvironment-mimicking matrix of S02 for culturing hematological malignant tumor organoids, the specific medium being composed of a basal medium and cytokines; the basal medium being a serum-free medium for hematopoietic stem cells; the cytokines including a first cytokine composition and specific cytokines; the first cytokine composition including penicillin / streptomycin, stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4; the specific cytokine being 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 interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4 is 1:1:1; S04: Identifying the immunophenotype of the cultured hematological malignant tumor organoids; screening out hematological malignant tumor organoids with an immunophenotype close to that of the patient's bone marrow cells.

[0008] As a preference, the tumor cell sample is a tumor cell sample derived from bone marrow.

[0009] Further, the hematological system malignant tumor is acute lymphoblastic leukemia or acute myeloid leukemia.

[0010] Further, the mass concentration of the GelMA solution is 5%-10%; the mass concentration of the photoinitiator is 0.01-0.05%.

[0011] As a preference, the mass concentration of the GelMA solution is 5%; the mass concentration of the photoinitiator is 0.05%.

[0012] Furthermore, in S02, 30,000 - 50,000 tumor cells are resuspended in every 25 μL of the bone marrow microenvironment-mimicking matrix solution. The bone marrow microenvironment-mimicking matrix provides an environment for the correct differentiation of the immunophenotype of cultured blood malignancy organoids.

[0013] As a preference, 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.

[0014] Furthermore, when the cultured blood malignancy is acute lymphoblastic leukemia, the specific cytokine added to the culture medium is interleukin-7; when the cultured blood malignancy is acute myeloid leukemia, the specific cytokine added to the culture medium is thrombopoietin.

[0015] Furthermore, the immunophenotype identification includes co-incubating the cultured blood malignancy organoids with blood malignancy-specific antibodies, and detecting the expression of the specific antibodies on the blood malignancy organoids; using the expression of the specific antibodies on the patient's bone marrow cells as a comparison.

[0016] As a preference, in the immunophenotype identification, the expression level of the specific antibodies on the blood malignancy organoids does not differ by more than 10% compared with that of the patient's bone marrow cells.

[0017] More preferably, the specific antibody is CD34.

[0018] More preferably, 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.

[0019] On the other hand, the present invention lies in providing an application of the cultured blood tumor organoids.

[0020] Use of the hematological malignancy organoids cultured by the above-mentioned culture method in preparing a preclinical drug resistance detection model.

[0021] Beneficial technical effects: The present invention first proposes a method for standardizing the construction of hematological system malignancy organoids. According to the method provided by the present invention, multiple cases of AML / ALL organoids have been cultured, proving that the method of the present invention has good repeatability.

[0022] Secondly, the hematological tumor organoids cultured in the present invention are derived from non-epithelial bone marrow cells and achieve rapid growth within 7 days of culture. Compared with the current method in the art that mostly uses epithelial-derived cells for culturing solid tumor organoids and has a longer culture period, the organoid culture method of the present invention has advantages. In addition, there are few reports on the successful culture of hematological tumor organoids at present, so the technical solution of the present invention has significant novelty.

[0023] Furthermore, the method system provided by the present invention not only successfully cultures hematological tumor organoids, but also further conducts phenotypic identification on them. The screened hematological tumor organoids are similar to the cellular immunophenotype of patients and can be used for preparing a drug resistance detection model in the future. The said drug resistance detection model can more accurately screen out which drugs are more sensitive to clinically drug-resistant patients, thereby increasing the therapeutic effect of the disease. Through experimental verification, the reason why the hematological tumor organoids prepared by the present invention can more accurately reflect the drug resistance situation is closely related to promoting the correct differentiation of the immunophenotype of the organoids in the preparation method.

[0024] Furthermore, the drug screening or drug sensitivity test results of the hematological tumor organoids cultured to be close to the cellular immunophenotype of patients can provide more accurate data support for the personalized precision treatment of patients.

[0025] Finally, the culture method provided by the present invention provides a set of innovative culture medium components and bone marrow microenvironment simulation matrix components. Among them, the culture medium uses the first cytokine composition with a streamlined but high concentration (specifically referring to several components such as stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4) and the addition of specific cytokines with a high concentration, which can efficiently culture hematological tumor organoids with a high survival rate. Further, by using the innovative bone marrow microenvironment simulation matrix components, the phenotypic differentiation of the organoids is more accurate compared with the commonly used Matrigel matrix in the art. Description of the Drawings

[0026] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following will briefly introduce the drawings required for the description of the embodiments or the prior art. In all the drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the elements or parts do not necessarily draw according to the actual scale. Obviously, the following described drawings are some embodiments of the present invention. For those of ordinary skill in the art, without creative efforts, other drawings can also be obtained according to these drawings.

[0027] Figure 1 Bright-field image of the growth of acute lymphoblastic leukemia organoids in one embodiment of the present invention (scale bar: 50 μm); Figure 2 Experimental results of the sensitivity of cultured ALL organoids to different anti-tumor drugs in one embodiment of the present invention; Figure 3 Bright-field image of the growth of acute myeloid leukemia under different culture conditions in one embodiment of the present invention; Figure 4 Experimental results of the drug sensitivity of different AML organoids to venetoclax in one embodiment of the present invention; Figure 5 Experimental results of the sensitivity of AML organoids in different culture systems to drugs in one embodiment of the present invention; Figure 6 Experimental results of the sensitivity of cultured AML organoids to different anti-tumor drugs in one embodiment of the present invention. Detailed implementation manners

[0028] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the following will clearly and completely describe the technical solutions in the embodiments of the present invention with reference to the drawings in the embodiments of the present invention. Obviously, the described embodiments are some but not all of the embodiments of the present invention. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative efforts belong to the scope of protection of the present invention.

[0029] As used herein, "and / or" includes any and all combinations of one or more of the listed related items.

[0030] As used herein, "a plurality of" means two or more, that is, it includes two, three, four, five, etc.

[0031] As used in this specification, the term "about" 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.

[0032] In this specification, certain embodiments may be disclosed in a format that is within a certain range. It should be understood that this description of "within a certain range" is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Accordingly, the description of a range should be considered to have specifically disclosed all possible sub-ranges and individual numerical values within that range. For example, the description of the range 1-6 should be considered to have specifically disclosed sub-ranges such as 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 within that range, such as 1, 2, 3, 4, 5, and 6. The above rules apply regardless of the breadth of the range.

[0033] Glossary of terms: "Close to the patient's immune phenotype" as used in the present invention means that the identification result by flow cytometry shows similarity to the immune phenotype (surface markers) of the patient's tumor cells.

[0034] Example 1 This example provides a method example for the culture, identification, and drug screening of hematological malignancy organoids.

[0035] 1. Method for culturing hematological tumor organoids.

[0036] S01: Take a bone marrow or peripheral blood sample from a patient, and obtain a 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, and add DPBS to terminate the lysis. Pre-cool the centrifuge in advance, centrifuge at 600 g and 4 °C for 5 min, and discard the supernatant. Then resuspend the cells in an appropriate amount of bone marrow microenvironment simulation matrix, and the bone marrow microenvironment simulation matrix is a 5% GelMA (containing 0.05% photoinitiator LAP) solution, seed it in a 48-well plate, and solidify.

[0037] S02: Add 200 μL of StemSpan SFEM II (serum-free medium for hematopoietic stem cells) and cytokines for culturing hematologic tumor organoids; 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 medium every 2 - 3 days, and passage the primary organoids obtained after culturing for 7 - 10 days. The obtained organoids are hematologic malignancy organoids, abbreviated as hematologic tumor organoids.

[0038] 2. Identification of hematologic tumor organoids.

[0039] Analyze the cell composition of organoids by flow cytometry and detect the expression of antibodies such as CD45 and CD34.

[0040] 3. Drug screening Take the successfully cultured hematologic tumor organoids above, digest them with digestive fluid and wash them with DPBS buffer, then centrifuge at 600 g for 5 min at 4 °C and discard the supernatant. Count the collected cells, resuspend them in medium, seed the cells at 1000 organoids / well in a 96-well plate, add the drug to be tested, and perform a cell viability test after 5 days.

[0041] Example 2 This example provides an example of constructing acute lymphoblastic leukemia (ALL) organoids.

[0042] 1. Primary culture: (1) The patient signs an informed consent form, take the patient's bone marrow sample, place it at room temperature for a period of time, after rinsing the pipette, dilute it with normal-temperature PBS.

[0043] (2) First add 15 mL of sample density gradient separation liquid into a 50 mL centrifuge tube, and then slowly add the diluted blood above the liquid level of the separation liquid.

[0044] (3) Centrifuge at 400 g for 30 minutes at room temperature.

[0045] (4)Remove the centrifuge tube from the centrifuge. At this time, the liquid level in the centrifuge tube is divided into four layers. From top to bottom, they are the first layer of plasma layer, the second layer of mononuclear cell layer, the third layer of separation liquid, and the fourth layer of red blood cells, etc. Discard the top layer, transfer the mononuclear cell layer to another centrifuge tube, add PBS with a volume one time that of the cells, and centrifuge at 300 g for 15 minutes at room temperature.

[0046] (5)After centrifugation, take the precipitate at the bottom, add 2 - 3 mL of red blood cell lysate, lyse on ice for 3 - 5 minutes, and centrifuge at 300 g for 15 minutes.

[0047] (6)After cell counting, take an appropriate number of cells, resuspend them in 5% GelMA (containing 0.05% photoinitiator LAP) and seed them in a 48 - well plate (resuspend 50,000 cells in 25 μL of 5% GelMA), cure them by irradiating with ultraviolet light for 60 s, add ALL organoid - specific medium for culture, and change the medium every 2 - 3 days. Passage every 7 - 10 days. The results of culturing for 7 days are shown in Figure 1 , and the results show that ALL organoids grow rapidly within 7 days.

[0048] ALL organoid - specific 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), 10 ng / ml BMP4 (bone morphogenetic protein 4), and 10 ng / ml IL - 7 (interleukin 7).

[0049] As a control, after cell counting, take an appropriate number of cells, resuspend them in Matrigel and seed them in a 48 - well plate (resuspend 50,000 cells in 25 μL of Matrigel), after it solidifies in the incubator, add ALL organoid - specific medium for culture, and change the medium every 2 - 3 days. Passage every 7 - 10 days.

[0050] 2. Sub - culture: (1)After the organoids grow to an appropriate number, perform digestion and amplification. Aspirate the medium in the cell culture plate, use a 1 mL pipette tip to scrape the organoid - GelMA mixed gel drops, collect them in a 15 mL centrifuge tube, pipette and mix the suspension evenly, and centrifuge at 300 g for 5 minutes.

[0051] (2)Discard the supernatant, add 2 - 3 mL of GelMA digestive solution (EFL) according to the volume of GelMA after centrifugation, and gently pipette 4 - 5 times with a pipette tip.

[0052] (3) Digest in a 37°C water bath. After complete digestion is observed under a microscope, terminate the digestion and centrifuge at 300 g for 5 min.

[0053] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5 min.

[0054] (5) Depending on the number of cell clusters, resuspend the cells in 5% GelMA and inoculate them in a 48-well plate. Irradiate with UV light for 60 seconds to solidify, add ALL organoid-specific culture medium, and replace the medium every 2-3 days. Subculture every 7-10 days.

[0055] 3. Cryopreservation: (1) After the organoids have grown to an appropriate number, they can be frozen. Aspirate the culture medium in the cell culture plate, aspirate PBS, use a 1 mL pipette to scrape the organoid-GelMA mixed gel droplets, collect them in a 15 mL centrifuge tube, use the pipette to blow and mix the suspension, and centrifuge at 300g for 5 min.

[0056] (2) Discard the supernatant and observe the amount of GelMA. Add 2-3 mL of organoid digestion solution according to the volume of GelMA after centrifugation, and gently blow 4-5 times with the pipette tip rinsed with rinse solution.

[0057] (3) Digest in a 37°C water bath. After complete digestion is observed under a microscope, terminate the digestion and centrifuge at 300 g for 5 min.

[0058] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5 min.

[0059] (5) Cryopreserved in liquid nitrogen.

[0060] 4. Resuscitation: (1) Remove the cryovials from liquid nitrogen and thaw them in a 37°C water bath.

[0061] (2) After thawing, transfer the organoid cryopreservation solution containing cells to a 50 mL centrifuge tube, slowly add 9 mL of pre-cooled PBS, and centrifuge at 600 g for 5 min.

[0062] (3) Discard the supernatant, transfer the cell pellet to a 15 mL centrifuge tube with PBS, and centrifuge at 600 g for 5 min.

[0063] (4) Discard the supernatant and resuspend the cells in 5% GelMA according to the number of cell clusters and inoculate them in a 48-well plate. Solidify them by UV light for 30 seconds and add ALL organoid-specific culture medium. Replace the medium every 2-3 days and subculture every 7-10 days.

[0064] 5. Identification: Flow cytometry: Take the bone marrow samples of the same patient and the single cells digested from the ALL organoids cultured in the above steps, and incubate them with the selected antibodies (CD34, HLA-DR, CD33, cD3, CD7, CD13) to specifically bind the antibodies to the antigens on the cell surface. Turn on the flow cytometer and perform preheating and calibration of the instrument; place the labeled samples in the flow cytometer for detection; the analysis and identification results are shown in Table 1.

[0065] Diagnostic criteria for ALL: When the proportion of blast cells (compared to nucleated cells) in the bone marrow is ≥ 20%, it is diagnosed as ALL. In this example, the marker of blast cells is specifically CD34. It should be noted that ALL can be divided into different subtypes, but in this example, the identification and typing of ALL are not performed, and only whether the cultured organoids are ALL is identified.

[0066] Table 1 Immunophenotypic analysis and identification of ALL organoids The results showed that the binding of ALL organoids cultured in 5% GelMA to the antibodies was almost the same as that of bone marrow cells of ALL patients, indicating that the phenotype of the cultured ALL organoids was very close to that of real patients. On the contrary, the binding of ALL organoids cultured in Matrigel matrix gel to the antibodies was quite different from that of bone marrow cells of ALL patients, indicating that the phenotype of ALL organoids cultured by this method was far from that of real patients.

[0067] 6. Drug screening: (1) When the above ALL organoids grow to a sufficient amount, collect the organoids, digest them into single cells, and count.

[0068] After centrifugation, discard the supernatant. According to the counting results, ensure that the seeding density is 500 - 1000 organoids / well.

[0069] (3) Prepare an appropriate amount of ALL organoid-specific medium in advance.

[0070] (4) Use a 1000 μL pipette to pipette and mix the cell suspension with 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.

[0071] (5) In the blank group, only 100 μL of cell-free medium is added to each well.

[0072] After seeding, PBS can be added to the surrounding wells to maintain humidity; place the 96-well plate in the incubator and wait for drug addition.

[0073] (7) Using a 200 μL pipette, add the prepared drug solutions of each target concentration of Venetoclax: 10, 20, 40, 100 (unit: μM); Vinblastine Sulfate: 0.001, 0.01, 0.1, 1, 10 (unit: nM) to the corresponding drug-added groups in sequence, 100 μL of the drug solution per well. Add the tip along the side wall of the well, and note to pipette several times before adding to mix the drug solution evenly.

[0074] (8) After aspirating the drug solutions of different concentrations, change the pipette tip for the next step.

[0075] (9) Only add 100 μL of the culture medium to the negative group and the blank group.

[0076] (10) After adding the drugs, gently tap the culture plate to mix the drug solution with the cells for a full reaction.

[0077] (11) Place the well plate in the incubator and perform ATP detection 5 days after the drug takes effect.

[0078] (12) The above-cultured ALL organoids are derived from the bone marrow cells of patients clinically resistant to Venetoclax. The drug detection results of the cultured ALL organoids are shown in Figure 2 , and the results show that this organoid model is more sensitive to Vinblastine Sulfate than Venetoclax and shows resistance to Venetoclax, which is consistent with the drug response of the patients.

[0079] Example 3 This example provides an example of the culture and drug screening of acute myeloid leukemia (AML) organoids.

[0080] 1. Culture of acute myeloid leukemia (AML) organoids: The method is the same as that in Example 2, and the AML organoid-specific culture medium is as follows.

[0081] AML organoid-specific culture 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), 10 ng / ml BMP4 (bone morphogenetic protein 4), and 25 ng / ml TPO (thrombopoietin).

[0082] AML organoid control medium: SFEM II medium (StemSpan SFEM II serum-free medium for hematopoietic stem cells) and specific cytokines; the specific cytokines consist 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).

[0083] 2. Different culture condition tests: AML organoids were cultured in a similar manner to Example 2, where a part of the AML organoids were cultured with the AML organoid control medium; another part of the AML organoids were cultured with the AML organoid specific medium; images were collected after 3 days of culture. The results are shown in Figure 3 , and it can be seen in the figure that the organoids in the group with added TPO grew faster and were larger in size.

[0084] On the other hand, a part of the AML organoids were resuspended in Matrigel and the AML organoid specific medium was added, and cultured for 3 days; a part of the AML organoids were resuspended in 5% GelMA and inoculated in a 48-well plate for photocuring, and the AML organoid specific medium was added, and cultured for 3 days. The results are shown in Figure 3 , and it can be seen in the figure that the organoid spheres in 5% GelMA were larger in size and more in number than those in Matrigel.

[0085] The immunophenotype of the cultured AML organoids was identified, and the organoids with an immunophenotype close to that of the bone marrow cells of the same patient were selected for subsequent experiments.

[0086] 3. Digestion of organoids: (1) When the organoids grew to a sufficient amount, the organoids were collected, digested into single cells, and counted.

[0087] (2) After centrifugation, the supernatant was discarded. According to the counting results, ensure that the seeding density was 500 - 1000 organoids per well.

[0088] 4. Cell seeding: (1) Prepare an appropriate amount of AML organoid specific medium in advance.

[0089] (2) Use a 1000 μL pipette to pipette and mix the cell suspension with adjusted density, and add it to a 96-well plate at a volume of 100 μL per well, trying to ensure that the number of organoids in each well was uniform.

[0090] (3) In the blank group, only 100 μL of cell-free medium was added to each well.

[0091] (4)After the plating is completed, PBS can be added to the surrounding wells to maintain humidity; place the 96-well plate in the incubator and wait for drug addition. The drugs used in this example are venetoclax, azacitidine, decitabine, and gilteritinib.

[0092] 5. Drug action (1)Using a 200 μL pipette, add the prepared target concentration drug solutions of venetoclax: 50, 5, 0.5, 0.05, 0.005, 0.0005, 0.00005 (unit: μM); azacitidine, decitabine, gilteritinib: 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 (unit: μM) to the corresponding drug addition groups in sequence, 100 μL of drug solution per well, add the tip along the side wall of the well, and note to pipette several times before addition to mix the drug solution evenly.

[0093] (2)After aspirating different concentration drug solutions, change the pipette tip for the next step.

[0094] (3)Only add 100 μL of culture medium to the negative group and the blank group.

[0095] (4)After drug addition, the culture plate can be gently tapped to mix the drug solution with the cells for sufficient reaction.

[0096] (5)Place the well plate in the incubator and perform detection after 5 days of drug action.

[0097] 6. Detection: After 5 days, perform ATP detection.

[0098] 7. Data analysis: According to the ATP detection results, use GraphPad Prism for data analysis. The experimental results are as Figure 4 shown. Among them, the AML organoid 1 model is derived from the bone marrow cells of patients without drug treatment; the AML organoid 2 model is derived from the bone marrow cells of patients clinically resistant to venetoclax. The in vitro drug sensitivity test results show that this model successfully retains the drug resistance characteristics of the original tumor. It is worth noting that different organoids show significant heterogeneity to the same drug, while the traditional cell line models (MOLM-13 and HL60) show high homogeneity in drug sensitivity response, proving that organoids have more advantages than traditional cell lines in simulating clinical individual differences.

[0099] Furthermore, to verify the influence of the microenvironment matrix on the drug resistance phenotype, the experiment further uses two culture systems of Matrigel and 5% GelMA hydrogel for comparative research ( Figure 5). Experiments show that: 1) In the experimental group supplemented with venetoclax, the AML organoids 2 cultured with Matrigel were sensitive to venetoclax, that is, the drug-resistant phenotype was reversed, indicating that the phenotype of the organoids shifted during the culture with Matrigel (consistent with the results of culturing ALL in Example 2), while the AML organoids 2 cultured with 5% GelMA hydrogel were resistant to venetoclax, consistent with the drug-resistant phenotype of the patients. 2) In the gilteritinib treatment group, the AML organoids 2 cultured with 5% GelMA hydrogel were significantly more sensitive to gilteritinib, demonstrating that an organoid model close to the patient phenotype can more accurately screen suitable therapeutic drugs for drug-resistant patients.

[0100] Furthermore, Figure 6 It shows the sensitivity detection of multiple drugs using patient-derived organoid models without prior drug treatment. The results show that the organoids have different responses to different drugs, that is, different sensitivities to different anti-tumor drugs. Since it has been confirmed in the previous experiments that the hematological tumor organoid model cultured by the method of the present invention has a phenotype closer to that of the patients, the results of preclinical drug screening tests using such hematological tumor organoids have high reference value.

[0101] It should be noted that in this article, the terms "including", "comprising" or any other variants thereof are intended to cover non-exclusive inclusion, so that a process, method, article or device including a series of elements not only includes those elements, but also includes other elements not explicitly listed, or elements inherent to such process, method, article or device. Without further limitations, an element defined by the statement "including one..." does not exclude the existence of additional identical elements in the process, method, article or device including that element.

[0102] The embodiments of the present invention have been described above in conjunction with the accompanying drawings, but the present invention is not limited to the above specific embodiments. The above specific embodiments are merely illustrative and not restrictive. Under the inspiration of the present invention, those of ordinary skill in the art can also make many forms without departing from the purpose of the present invention and the scope protected by the claims. All of these are within the protection scope of the present invention.

Claims

1. A method for culturing blood malignant tumor organoids with an immune phenotype close to that of a patient, characterized in that, It includes the following steps: S01: Take cell samples of patients with hematological malignancies, and the cell samples include tumor cell samples derived from bone marrow and / or tumor cell samples derived from peripheral blood; S02: Prepare a bone marrow microenvironment-mimicking matrix, which is a methacrylated gelatin solution containing a photoinitiator. Resuspend the tumor cell samples in the bone marrow microenvironment-mimicking matrix solution and perform ultraviolet light curing; S03: Add a specific medium to the bone marrow microenvironment-mimicking matrix in S02 for culturing hematological malignancy organoids. The specific medium is composed of a basal medium and cytokines; the basal medium is a serum-free medium for hematopoietic stem cells; the cytokines include a first cytokine composition and specific cytokines; 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 interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4 is 1:1:1; S04: Perform immunophenotypic identification on the cultured hematological malignancy organoids; screen out hematological malignancy organoids with an immunophenotype close to that of the patient's bone marrow cells.

2. The cultivation method according to claim 1, characterized in that, The mass concentration of the methacrylated gelatin solution is 5%-10%; the mass concentration of the photoinitiator is 0.01%-0.05%.

3. The culturing method according to claim 1, wherein In S02, 30,000-50,000 tumor cells are resuspended in each 25 μL of the bone marrow microenvironment-mimicking matrix solution.

4. The cultivation method according to claim 1, characterized in that The first cytokine composition is specifically composed 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.

5. The culturing method according to claim 1, wherein, When the cultured hematological malignancy is acute lymphoblastic leukemia, the specific cytokine added to the medium is interleukin 7.

6. The cultivation method according to claim 1, characterized in that, When the cultured hematological malignancy is acute myeloid leukemia, the specific cytokine added to the medium is thrombopoietin.

7. The cultivation method according to claim 1, characterized in that, The immunophenotypic identification includes co-incubating the cultured hematological malignancy organoids with hematological malignancy-specific antibodies and detecting the expression of the specific antibodies on the hematological malignancy organoids; using the expression of the specific antibodies on the patient's bone marrow cells as a comparison.

8. The culturing method according to claim 7, wherein, In the immunophenotypic identification, the expression level of the specific antibody on the hematological malignancy organoids does not differ by more than 10% compared with that of the patient's bone marrow cells.

9. The cultivation method according to claim 5, characterized in that, 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.

10. Use of the hematological malignancy organoids obtained by the culture method according to any one of claims 1-9 in the preparation of a preclinical drug resistance detection model.

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