Culture reagent composition and culture method for organoids from hematologic malignancies

Abstract

This invention belongs to the field of biomedical technology, specifically relating to a culture reagent composition and method for organoids from hematologic malignancies. The invention first provides a reagent composition for culturing organoids from hematologic malignancies, including acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition includes a bone marrow microenvironment mimicking matrix reagent and a specific culture medium. Using the reagent composition of this invention, hematologic malignancies can be cultured efficiently with high survival rates. Furthermore, the bone marrow microenvironment mimicking matrix component used in this invention, compared to the commonly used Matrigel matrix in the field, allows for more accurate phenotypic differentiation of hematologic malignancies.

Description

[0001] Divisional application

[0002] This application is a divisional application of Chinese Patent Application No. 2025107269727, filed on June 3, 2025, entitled "Method and Application of Organoid Culture for Malignant Tumors of the Hematopoietic System". Technical Field

[0003] This invention belongs to the field of biomedical technology, specifically relating to a culture reagent composition and culture method for organoids of hematologic malignancies. Background Technology

[0004] Hematologic malignancies refer to malignant tumors occurring in the blood system (bone marrow, hematopoietic tissue, and lymphoid tissue), mainly including leukemia, lymphoma, and multiple myeloma. Leukemia and lymphoma are both among the top ten causes of cancer incidence and mortality worldwide. Currently, hematologic malignancies are classified according to morphology, immunophenotype, cytogenetic and molecular abnormalities, and clinical characteristics, primarily into myeloid or lymphoid malignancies, and acute or chronic forms. Leukemia and lymphoma share many similarities. When tumor cells (lymphoblasts) affect the blood and bone marrow (defined as more than 20% of bone marrow blast cells), the disease can manifest as leukemia; when blast cells primarily infiltrate extramedullary tissues to form tissue masses, the disease can manifest as lymphoma.

[0005] Patient-derived cell culture 3D models have been reported for various solid tumors, but reports on 3D models of hematologic malignancies (such as leukemia and lymphoma) are rare. While some literature reports the use of iPSC-derived or healthy donor hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) to construct bone marrow organoids to mimic the normal bone marrow microenvironment, constructing organoids for hematologic malignancies is significantly more challenging. The construction of hematologic malignancies often requires overcoming key challenges such as spontaneous differentiation / apoptosis of malignant cells, clonal selection bias, and insufficient biomimicry of the microenvironment. Furthermore, hematologic malignancies are prone to death or heterodifferentiation during culture, which can lead to discrepancies between drug sensitivity test results and clinical responses, resulting in inaccurate drug testing results based on organoid models.

[0006] In conclusion, there is an urgent need to propose new methods and strategies to improve upon the shortcomings of the existing technologies. Summary of the Invention

[0007] The purpose of this invention is to provide a culture reagent composition and culture method for organoids of hematologic malignancies, which partially solves or alleviates the above-mentioned deficiencies in the prior art. The specific technical solution adopted by this invention is as follows.

[0008] A first aspect of the present invention is to provide a composition for culturing organoids of hematologic malignancies.

[0009] A reagent composition for culturing organoids from hematologic malignancies, including acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition comprises a bone marrow microenvironment mimicry matrix reagent and a specific culture medium;

[0010] The bone marrow microenvironment mimicry matrix reagent provides an environment for the correct differentiation of cultured hematologic malignancy organoids into immunophenotypes.

[0011] The bone marrow microenvironment simulation matrix reagent is a solution of methacrylamide gelatin (GelMA) containing a photoinitiator;

[0012] The specific culture medium consists of a basal culture medium and cytokines; the basal culture medium is a serum-free culture 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 cytokines are 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.

[0013] The bone marrow microenvironment simulation matrix reagent and the specific culture medium are set up independently. When used for organoid culture of hematologic malignancies, tumor cells are first added to the bone marrow microenvironment simulation matrix reagent for photopolymerization, and then the specific culture medium is added.

[0014] When the tumor cells are acute lymphoblastic leukemia cells, the specific cytokine is interleukin-7;

[0015] When the tumor cells are acute myeloid leukemia cells, the specific cytokine is thrombopoietin.

[0016] Furthermore, the mass concentration of the methacrylamide gelatin solution is 5%-10%; and the mass concentration of the photoinitiator is 0.01%-0.05%.

[0017] As a preferred embodiment, the photoinitiator is a photoinitiator capable of initiating ultraviolet curing; specifically, the photoinitiator is lithium phenyl (2,4,6-trimethylbenzoyl) phosphate, but this is not a limitation.

[0018] As a preferred embodiment, the mass concentration of the methacrylamide gelatin solution is 5%; and the mass concentration of the photoinitiator is 0.05%.

[0019] Furthermore, the first cytokine composition specifically comprises 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.

[0020] As a preferred embodiment, the first cytokine composition specifically comprises 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.

[0021] Another aspect of the present invention is to provide a method for culturing organoids from hematological malignancies.

[0022] A method for culturing hematologic malignancy organoids with an immunophenotype closely similar to that of a patient, based on the above-described reagent composition for culturing hematologic malignancy organoids, includes the following steps:

[0023] S01: Take tumor cell samples, including acute lymphoblastic leukemia cell samples or acute myeloid leukemia cell samples, add them to the bone marrow microenvironment simulation matrix reagent of the reagent composition for resuspending, and cure under ultraviolet light; 30,000-50,000 tumor cell samples are resuspended in each 25 μL of the bone marrow microenvironment simulation matrix reagent;

[0024] S02: Add a specific culture medium to the bone marrow microenvironment mimic matrix reagent containing tumor cell samples for the culture of organoids of hematologic malignancies;

[0025] S03: Identify the immunophenotype of the cultured hematologic malignant tumor organoids; screen out hematologic malignant tumor organoids that are similar to the immunophenotype of the patient's bone marrow cells.

[0026] Furthermore, the tumor cell sample is a tumor cell sample derived from bone marrow or a tumor cell sample derived from peripheral blood.

[0027] Furthermore, the immunophenotypic identification includes co-incubating the cultured hematologic malignancy organoids with hematologic malignancy-specific antibodies, detecting the expression of the specific antibodies on the hematologic malignancy organoids, and using the expression of the specific antibodies on the patient's bone marrow cells as a comparison;

[0028] The expression level of the specific antibody on the organoids of the hematologic malignancies does not differ by more than 10% compared with that on the patient's bone marrow cells.

[0029] Furthermore, the specific antibody includes CD34.

[0030] Application of hematologic malignant tumor organoids cultured by the above method in the preparation of preclinical drug resistance detection models.

[0031] Beneficial technical effects:

[0032] This invention first provides a culture reagent composition for culturing organoids from hematologic malignancies (mainly acute lymphoblastic leukemia or acute myeloid leukemia). This composition includes innovative culture medium components and a bone marrow microenvironment-mimicking matrix component. The culture medium employs a simplified but high-concentration composition of first-order cytokines (specifically stem cell factor, FMS-like tyrosine kinase 3, interleukin 3, fibroblast growth factor 2, and bone morphogenetic protein 4) and high concentrations of specific cytokines, enabling efficient culture of hematologic malignancies with high survival rates. Furthermore, the innovative bone marrow microenvironment-mimicking matrix component, compared to the commonly used Matrigel matrix in the art, allows for more accurate phenotypic differentiation of hematologic malignancies.

[0033] Secondly, based on the above-mentioned culture reagent composition, this invention also provides a standardized method for constructing organoids from hematologic malignancies. Multiple AML / ALL organoids have been cultured using this method, demonstrating its excellent reproducibility. This invention uses non-epithelial bone marrow cells to culture hematologic tumor organoids, achieving rapid growth within 7 days of culture. Compared to current practices in the field, which often use epithelial cells and have longer culture periods for solid tumor organoids, this invention's organoid culture method has significant advantages. Furthermore, successful reports of hematologic tumor organoid culture are rare; therefore, this invention possesses significant novelty.

[0034] Finally, the present invention also performed phenotypic identification on the cultured hematologic tumor organoids. The screened hematologic tumor organoids are similar to the cellular immunophenotype of patients and can be used to prepare drug resistance detection models. These drug resistance detection models can more accurately screen which drugs clinically resistant patients are more sensitive to, thereby increasing the effectiveness of disease treatment. Experimental verification shows that the reason why the hematologic tumor organoids prepared by this invention can more accurately reflect drug resistance is closely related to the presence of reagents in the composition provided by this invention that promote the correct differentiation of the immunophenotype of hematologic tumor organoids. Attached Figure Description

[0035] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the drawings used in the description of the embodiments or the prior art will be briefly introduced below. In all the drawings, similar elements or parts are generally identified by similar reference numerals. The elements or parts in the drawings are not necessarily drawn to scale. Obviously, the drawings described below are some embodiments of the present invention, and those skilled in the art can obtain other drawings based on these drawings without any creative effort.

[0036] Figure 1 This is a bright field image (scale bar 50 μm) of the growth of organoids in acute lymphoblastic leukemia in one embodiment of the present invention.

[0037] Figure 2 The results of the sensitivity experiment of cultured ALL organoids to different antitumor drugs in one embodiment of the present invention are shown.

[0038] Figure 3 This is a bright field diagram of the growth of acute myeloid leukemia under different culture conditions in one embodiment of the present invention;

[0039] Figure 4 The results of drug sensitivity experiments on veneclade for different AML organoids are shown in one embodiment of the present invention.

[0040] Figure 5 The results of drug sensitivity experiments on AML organoids in different culture systems in one embodiment of the present invention are shown.

[0041] Figure 6 This is the result of an experiment on the sensitivity of cultured AML organoids to different antitumor drugs in one embodiment of the present invention. Detailed Implementation

[0042] To make the objectives, technical solutions, and advantages of the embodiments of the present invention clearer, the technical solutions of the embodiments of the present invention will be clearly and completely described below with reference to the accompanying drawings. Obviously, the described embodiments are only some, not all, of the embodiments of the present invention. All other embodiments obtained by those skilled in the art based on the embodiments of the present invention without creative effort are within the scope of protection of the present invention.

[0043] In this document, "and / or" includes any and all combinations of one or more of the listed related items.

[0044] In this article, "multiple" means two or more, that is, it includes two, three, four, five, etc.

[0045] As used in this specification, the term "about" typically means + / -5% of the value, more typically + / -4% of the value, more typically + / -3% of the value, more typically + / -2% of the value, even more typically + / -1% of the value, and even more typically + / -0.5% of the value.

[0046] In this specification, certain embodiments may be disclosed in a range-bound format. It should be understood that this "range-bound" description is merely for convenience and brevity and should not be construed as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered as having specifically disclosed all possible subranges and the individual numerical values ​​within those ranges. For example, a description of the range 1-6 should be considered as having specifically disclosed subranges 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., and the individual numbers within those ranges, such as 1, 2, 3, 4, 5, and 6. This rule applies regardless of the breadth of the range.

[0047] Definition of noun:

[0048] The phrase "similar to the patient's immune phenotype" as used in this invention refers to the similarity between the immune phenotype (surface markers) of the patient's tumor cells as determined by flow cytometry.

[0049] Example 1

[0050] This embodiment provides an example of a method for culturing, identifying, and screening drugs for organoids from hematologic malignancies.

[0051] 1. Culture methods for hematologic tumor organoids.

[0052] S01: Obtain a bone marrow or peripheral blood sample from the patient, and obtain a tumor cell sample after density gradient centrifugation. Resuspend the tumor cell sample in an appropriate amount of erythrocyte lysis buffer, lyse on ice for 3 min, and add DPBS to terminate the lysis. Pre-cool the centrifuge beforehand, centrifuge at 600 g, 4 ℃ for 5 min, and discard the supernatant. Resuspend the cells in an appropriate amount of bone marrow microenvironment simulation matrix, which is a 5% GelMA solution (containing 0.05% photoinitiator LAP), seed in 48-well plates, and solidify.

[0053] S02: Add 200 μL of SFEM II medium (StemSpan SFEM II serum-free hematopoietic stem cell medium) and cytokines for hematologic malignancy 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). Change the organoid medium every 2-3 days. After 7-10 days of culture, passage the obtained primary organoids. The resulting organoids are hematologic malignancy organoids, or hematologic malignancy organoids for short.

[0054] 2. Identification of organoids from hematologic tumors.

[0055] Flow cytometry was used to analyze the composition of organoid cells and detect the expression of antibodies such as CD45 and CD34.

[0056] 3. Drug screening.

[0057] Collect the successfully cultured hematologic tumor organoids, digest them with digestive fluid, wash them with DPBS buffer, centrifuge at 600g, 4℃ for 5 min, and discard the supernatant. Count the collected cells, resuspend them in culture medium, and seed them at a rate of 1000 organoids / well in 96-well plates. Add the test drug, and perform cell viability testing after 5 days.

[0058] Example 2

[0059] This embodiment provides an example of organoid construction for acute lymphoblastic leukemia (ALL).

[0060] 1. Primary culture:

[0061] (1) The patient signed the informed consent form, the patient's bone marrow sample was taken, placed at room temperature for a period of time, the pipette was rinsed and then diluted with room temperature PBS.

[0062] (2) First add 15 mL of sample density gradient separation solution to a 50 mL centrifuge tube, and then slowly add diluted blood to the surface of the separation solution.

[0063] (3) Centrifuge at 400 g for 30 minutes at room temperature.

[0064] (4) Remove the centrifuge tube from the centrifuge. At this time, the liquid surface of the centrifuge tube is divided into four layers, from top to bottom: the first layer is plasma, the second layer is mononuclear cell, the third layer is separation solution, and the fourth layer is red blood cells. Discard the top layer, 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.

[0065] (5) After centrifugation, take the bottom precipitate, add 2-3 mL of red blood cell lysis buffer, lyse on ice for 3-5 minutes, and centrifuge at 300 g for 15 minutes.

[0066] (6) After cell counting, take an appropriate number of cells, resuspend them in 5% GelMA (containing 0.05% photoinitiator LAP), and seed them in 48-well plates (25 μL of 5% GelMA to resuspend 50,000 cells). Irradiate with UV light for 60 seconds to solidify, add ALL organoid-specific culture medium, and change the medium every 2-3 days. Passage every 7-10 days. The results after 7 days of culture are shown in [the table below]. Figure 1 The results showed that ALL organoids grew faster over a 7-day period.

[0067] ALL organoid specific culture medium: SFEM II medium (StemSpan SFEM II serum-free hematopoietic stem cell medium) 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), 10 ng / mL BMP4 (bone morphogenetic protein 4) and 10 ng / mL IL-7 (interleukin 7).

[0068] As a control, after cell counting, an appropriate number of cells were taken, resuspended in Matrigel, and seeded in 48-well plates (50,000 cells resuspended in 25 μL Matrigel). After solidification in an incubator, ALL organoid-specific culture medium was added and cultured, with the medium changed every 2-3 days. The cells were passaged every 7-10 days.

[0069] 2. Passage breeding:

[0070] (1) After the organoids have grown to a suitable number, digestion and expansion are performed. The culture medium in the cell culture plate is removed, and the organoid-GelMA mixed droplets are scraped with a 1mL pipette tip and collected in a 15mL centrifuge tube. The suspension is mixed by pipetting and centrifuging at 300g for 5min.

[0071] (2) Discard the supernatant, add 2-3 mL of GelMA digestion solution (EFL) according to the volume of GelMA after centrifugation, and gently blow it 4-5 times with the pipette tip.

[0072] (3) Place it in a 37°C water bath for digestion. After complete digestion is observed under a microscope, stop digestion and centrifuge at 300g for 5 minutes.

[0073] (4) Discard the supernatant, resuspend the precipitate in PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5min.

[0074] (5) Based on the number of cell clusters, resuspend the cells in 5% GelMA and seed them in 48-well plates. Irradiate with UV light for 60 seconds to solidify the cells, add ALL organoid specific culture medium, and change the medium every 2-3 days. Passage the cells every 7-10 days.

[0075] 3. Cryopreservation:

[0076] (1) After the organoids have grown to a suitable number, they are frozen. The culture medium in the cell culture plate is removed, PBS is taken out, and organoid-GelMA mixed droplets are scraped with a 1mL pipette tip and collected in a 15mL centrifuge tube. The suspension is mixed by pipetting and centrifuging at 300g for 5min.

[0077] (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 the pipette tip rinsed with rinsing solution 4-5 times.

[0078] (3) Place it in a 37°C water bath for digestion. After complete digestion is observed under a microscope, stop digestion and centrifuge at 300g for 5 minutes.

[0079] (4) Discard the supernatant, resuspend the precipitate in PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5min.

[0080] (5) Store in liquid nitrogen.

[0081] 4. Recovery:

[0082] (1) Remove the cryovial from the liquid nitrogen and thaw it in a water bath at 37°C.

[0083] (2) After thawing, transfer the organoid cryopreservation solution containing cells to a 50mL centrifuge tube, slowly add 9mL of pre-cooled PBS, and centrifuge at 600g for 5min.

[0084] (3) Discard the supernatant, transfer the cell pellet to a 15mL centrifuge tube with PBS, and centrifuge at 600g for 5min.

[0085] (4) Discard the supernatant, and according to the number of cell clusters, resuspend the cells in 5% GelMA and seed them in 48-well plates. Irradiate with UV light for 30 seconds to solidify the cells, add ALL organoid specific culture medium, and change the medium every 2-3 days. Passage the cells every 7-10 days.

[0086] 5. Identification:

[0087] Flow cytometry: Bone marrow samples from the same patient and single cells digested from ALL-type organs cultured in the above steps were incubated with selected antibodies (CD34, HLA-DR, CD33, cD3, CD7, CD13) to allow the antibodies to specifically bind to the antigens on the cell surface. The flow cytometer was turned on for preheating and calibration; the labeled samples were placed in the flow cytometer for analysis; the analysis and identification results are shown in Table 1.

[0088] Diagnostic criteria for ALL: A bone marrow blast cell (ratio of nucleated cells) of ≥20% is diagnostic of ALL. In this embodiment, the specific marker for blast cells is CD34. It should be noted that ALL can be divided into different subtypes, but this embodiment does not perform ALL identification and subtyping, but only identifies whether the cultured organoids are ALL.

[0089] Table 1. Immunophenotypic analysis and identification of ALL organoids

[0090]

[0091] The results showed that the antibody binding of ALL organoids cultured in 5% GelMA was almost identical to that of bone marrow cells from ALL patients, indicating that the phenotype of the cultured ALL organoids was very close to that of real patients. Conversely, the antibody binding of ALL organoids cultured in Matrigel differed significantly from that of bone marrow cells from ALL patients, indicating that the phenotype of ALL organoids cultured by this method was far removed from that of real patients.

[0092] 6. Drug screening:

[0093] (1) When the above ALL organoids grow to a sufficient quantity, collect the organoids, digest them into single cells, and count them.

[0094] (2) After centrifugation, discard the supernatant and ensure that the plate density is 500-1000 organoids per well based on the counting results.

[0095] (3) Prepare an appropriate amount of ALL organoid specific culture medium in advance.

[0096] (4) Using a 1000 μL pipette, mix the adjusted cell suspension by pipetting 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.

[0097] (5) Add 100 μL of cell-free culture medium to each well of the blank group.

[0098] (6) 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 drug to be added.

[0099] (7) Using a 200μL pipette, add the prepared target concentrations of Venecra: 10, 20, 40, 100 (unit μM); and vinorelbine sulfate: 0.001, 0.01, 0.1, 1, 10 (unit nM) to the corresponding dosing groups in sequence, 100μL of drug solution per well, with the pipette tip added along the side wall of the well. Note that the pipette should be blown several times before adding to mix the drug solution.

[0100] (8) After drawing different concentrations of medicine, change the nozzle to proceed to the next step.

[0101] (9) Only 100 μL of culture medium was added to the negative group and the blank group.

[0102] (10) After the drug is added, you can gently tap the culture plate to mix the drug solution with the cells for a full reaction.

[0103] (11) Place the well plate in an incubator and perform ATP detection 5 days after drug treatment.

[0104] (12) The ALL organoids cultured above were derived from bone marrow cells of patients resistant to veneclade in clinical practice. The drug test results of the cultured ALL organoids are shown in […]. Figure 2 The results showed that the organoid model was more sensitive to vindesine sulfate than to veneclade and exhibited resistance to veneclade, which was consistent with the patient's response to the drug.

[0105] Example 3

[0106] This embodiment provides an example of organoid culture and drug screening for acute myeloid leukemia (AML).

[0107] 1. Culture of organoids for acute myeloid leukemia (AML): The method is the same as in Example 2, and the AML organoid-specific culture medium used is as follows.

[0108] AML organoid specific culture medium: SFEM II medium (StemSpan SFEM II serum-free hematopoietic stem cell medium) 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 LFGF2 (fibroblast growth factor 2), 10 ng / mL BMP4 (bone morphogenetic protein 4) and 25 ng / mL TPO (thrombopoietin).

[0109] AML organoid control culture medium: SFEM II medium (StemSpan SFEM II serum-free hematopoietic stem cell medium) 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 LFGF2 (fibroblast growth factor 2) and 10 ng / mL BMP4 (bone morphogenetic protein 4).

[0110] 2. Testing under different culture conditions: AML organoids were cultured using a method similar to that in Example 2, with one portion of the AML organoids cultured in AML organoid control medium and the other portion in AML organoid-specific medium. Images were acquired after 3 days of culture. Results are shown below. Figure 3 As can be seen in the figure, the organoids in the TPO group grew faster and were larger.

[0111] On the other hand, a portion of the AML organoids were resuspended in Matrigel and cultured for 3 days with AML organoid-specific medium; another portion of the AML organoids were resuspended in 5% GelMA and seeded in 48-well plates for photocuring, with AML organoid-specific medium added, and cultured for 3 days. Results are shown below. Figure 3 As can be seen in the figure, the organoid spheres in 5% GelMA are larger and more numerous than those in the matrix gel.

[0112] Immunophenotypic identification was performed on the cultured AML organoids, and organoids with immunophenotypes similar to those of bone marrow cells from the same patient were screened for subsequent experiments.

[0113] 3. Organoid digestion:

[0114] (1) When the organoids grow to a sufficient quantity, collect the organoids, digest them into single cells, and count them.

[0115] (2) After centrifugation, discard the supernatant and ensure that the plate density is 500-1000 organoids per well based on the counting results.

[0116] 4. Cell plating:

[0117] (1) Prepare an appropriate amount of AML organoid-specific culture medium in advance.

[0118] (2) Using a 1000μL pipette, mix the cell suspension after adjusting the density by pipetting, and add it to the 96-well plate at a volume of 100μL / well, so as to ensure that the number of organoids in each well is as uniform as possible.

[0119] (3) Add 100 μL of cell-free culture medium to each well of the blank group.

[0120] (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.

[0121] 5. Drug effects:

[0122] (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.

[0123] (2) After drawing up different concentrations of drug solution, change the nozzle to proceed to the next step.

[0124] (3) Only 100 μL of culture medium was added to the negative group and the blank group.

[0125] (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.

[0126] (5) Place the well plate in an incubator and perform the test after 5 days of drug treatment.

[0127] 6. Testing:

[0128] Five days later, an ATP test was performed.

[0129] 7. Data Analysis:

[0130] Based on the ATP detection results, data analysis was performed using GraphPad Prism, and the experimental results are as follows: Figure 4As 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.

[0131] 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 The experiments showed that: 1) In the group treated with veneclade, the AML organoid 2 cultured on Matrigel was sensitive to veneclade, indicating a reversal of the drug resistance phenotype. This suggests that the organoid phenotype shifted during the Matrigel culture process (consistent with the results of ALL cultured in Example 2). The AML organoid 2 cultured on 5% GelMA hydrogel was resistant to veneclade, consistent with the patient's drug resistance phenotype. 2) In the gilteritinib treatment group, the AML organoid 2 cultured on 5% GelMA hydrogel showed significantly stronger sensitivity to gilteritinib, demonstrating that organoid models with phenotypes closely resembling those of patients can more accurately screen suitable therapeutic agents for drug-resistant patients.

[0132] further, Figure 6 The study demonstrated the sensitivity testing of various drugs using organoid models derived from patients who had not undergone drug treatment. The results showed that these organoids exhibited different responses to different drugs, demonstrating varying sensitivities to different antitumor agents. Since previous experiments have confirmed that the hematologic tumor organoid models cultured using the method of this invention have a phenotype more closely resemble that of patients, the results of preclinical drug screening trials using such hematologic tumor organoids are highly reliable.

[0133] It should be noted that, in this document, the terms "comprising," "including," or any other variations thereof are intended to cover non-exclusive inclusion, such that a process, method, article, or apparatus that comprises a list of elements includes not only those elements but also other elements not expressly listed, or elements inherent to such a process, method, article, or apparatus. Unless otherwise specified, an element defined by the phrase "comprising one..." does not exclude the presence of other identical elements in the process, method, article, or apparatus that includes that element.

[0134] The embodiments of the present invention have been described above with reference to the accompanying drawings. However, the present invention is not limited to the specific embodiments described above. The specific embodiments described above are merely illustrative and not restrictive. Those skilled in the art can make many other forms under the guidance of the present invention without departing from the spirit and scope of the claims. All of these forms are within the protection scope of the present invention.

Claims

1. A reagent composition for hematological malignancy organoid culture, characterized by, The blood malignancy is acute lymphoblastic leukemia or acute myeloid leukemia; the reagent composition comprises 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 mass concentration of the methacrylated gelatin solution is 5%; the mass concentration of the photoinitiator is 0.05%; 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 comprises a first cytokine composition and a specific cytokine; The first cytokine composition comprises 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 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 into 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 photoinitiator is a lithium salt of phenyl(2,4,6-trimethylbenzoyl)phosphine oxide.

3. The reagent composition according to 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.

4. The reagent composition according to claim 3, 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.

5. The method of culturing a blood malignancy organoid having a close immune phenotype to a patient based on the reagent composition of any one of claims 1-4, characterized in that, The method comprises the following steps: S01: Take a tumor cell sample, which is an acute lymphoblastic leukemia cell sample or an acute myeloid leukemia cell sample, resuspend it in the bone marrow microenvironment simulation matrix reagent of the reagent composition, and photocure it under ultraviolet light; resuspend 30000-50000 tumor cell samples in each 25 μL of the bone marrow microenvironment mimetic matrix reagent; S02: adding specific medium into the bone marrow microenvironment mimetic matrix reagent containing the tumor cell samples for blood malignant tumor organoid culture; S03: immunophenotypic identification of the blood malignant tumor organoids obtained by culture; screening out blood malignant tumor organoids close to the immunophenotype of the patient's bone marrow cells.

6. The method of claim 5, wherein, The tumor cell samples are tumor cell samples derived from bone marrow or tumor cell samples derived from peripheral blood.

7. The method of claim 5, wherein, The immunophenotypic identification comprises co-incubating the blood malignant tumor organoids obtained by culture with blood malignant tumor specific antibodies, detecting the expression of the specific antibodies on the blood malignant tumor organoids; taking the expression of the specific antibodies on the patient's bone marrow cells as a comparison; The expression amount of the specific antibodies on the blood malignant tumor organoids differs by no more than 10% compared with the patient's bone marrow cells.

8. The method of claim 7, wherein, The specific antibodies include CD34.

Images