Culture methods and applications of hematologic malignancies organoids

Abstract

The present invention belongs to the field of biomedicine technology, and specifically relates to a method and application for culturing organoids of hematological malignancies. The culturing method of the present invention comprises: S01: obtaining a tumor cell sample; S02: preparing a bone marrow microenvironment-simulating matrix and resuspending the tumor cells; S03: adding a specific culture medium to culture hematological malignant tumor organoids; S04: phenotypic identification of the cultured hematological malignant tumor organoids; and screening out hematological malignant tumor organoids that are close to the patient's bone marrow cell phenotype. The method system of the present invention not only successfully cultured hematological malignant tumor organoids, but also further performed phenotypic identification on them. The screened hematological malignant tumor organoids are close to the patient's cell immune phenotype and can be used for accurate detection of clinical patient drug sensitivity and effective testing of potentially effective target drugs.

Description

Technical Field

[0001] The present invention belongs to the field of biomedicine technology, and specifically relates to a method for culturing organoids of malignant tumors of the blood system and its application. Background Art

[0002] Hematologic malignancies refer to malignancies that arise in the blood system (bone marrow, hematopoietic tissue, and lymphoid tissue), primarily including leukemia, lymphoma, and multiple myeloma. Leukemia and lymphoma rank among the top ten cancers in terms of morbidity and mortality worldwide. Hematologic malignancies are currently classified based on morphology, immunophenotype, cytogenetic and molecular abnormalities, and clinical features, primarily as myeloid or lymphoid, and acute or chronic. Leukemia and lymphoma share many similarities. When tumor cells (lymphoblasts) invade the blood and bone marrow (defined as greater than 20% of bone marrow blasts), the disease can present as leukemia; when blasts primarily infiltrate extramedullary tissues, forming tissue masses, the disease can present as lymphoma.

[0003] While patient-derived cell culture 3D models have been reported for a variety of solid tumors, reports of 3D models for hematological malignancies (such as leukemia and lymphoma) are rare. While there have been reports of bone marrow organoids generated from iPSCs or healthy donors using hematopoietic stem cells (HSCs) or mesenchymal stem cells (MSCs) to mimic the normal bone marrow microenvironment, the generation of organoids for hematological malignancies is more challenging. The generation of organoids for hematological malignancies often requires overcoming key challenges such as spontaneous differentiation / apoptosis of malignant cells, clonal selection bias, and insufficient microenvironmental resemblance. Specifically, hematological malignancy cells are prone to spontaneous differentiation or apoptosis during in vitro culture due to isolation from their native microenvironment, resulting in organoids that fail to faithfully recapitulate the molecular characteristics of patient tumors. Furthermore, because hematological malignancies exhibit high clonal heterogeneity, conventional culture media can induce clonal selection bias, whereby dominant clones proliferate while rare, drug-resistant subpopulations or cancer stem cells are gradually eliminated, reducing the predictive value of the generated models for recurrence mechanisms. Furthermore, hematologic malignancy organoids are prone to death or heterodifferentiation during culture, which can lead to deviations between drug sensitivity test results and clinical responses, necessitating precise control of the culture system. Furthermore, unlike bone marrow organoids, the cells used to construct hematologic malignancy organoids include patient-derived bone marrow or peripheral blood cells. During culture, the proliferation, heterogeneity, and pathological characteristics of malignant clones must be specifically maintained in a three-dimensional system. Furthermore, the tumor-associated stromal cells, extracellular matrix, and abnormal cytokine networks must be simulated, which increases the difficulty of construction.

[0004] In summary, there is an urgent need to propose a standardized method for constructing organoids of hematological malignancies to supplement the shortcomings of existing technologies. Summary of the Invention

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

[0006] The first aspect of the present invention is to provide a method for culturing organoids of hematological malignancies.

[0007] A method for culturing hematological malignancy organoids having an immunophenotype close to that of a patient, comprising the following steps:

[0008] S01: Obtain a cell sample from a patient with a hematological malignancy, wherein the cell sample includes a tumor cell sample derived from bone marrow and / or a tumor cell sample derived from peripheral blood;

[0009] S02: preparing a bone marrow microenvironment simulation matrix, wherein the bone marrow microenvironment simulation matrix is ​​a methacrylated gelatin (GelMA) solution containing a photoinitiator, and resuspending the tumor cells in the bone marrow microenvironment simulation matrix solution for UV curing;

[0010] S03: adding a specific culture medium to the bone marrow microenvironment-simulating matrix of S02 to culture hematological malignancy organoids, wherein the specific culture medium is composed 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 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;

[0011] S04: Perform immunophenotyping on the cultured hematological malignancy organoids; screen out hematological malignancy organoids that are close to the patient's bone marrow cell phenotype.

[0012] Preferably, the tumor cell sample is a tumor cell sample derived from bone marrow.

[0013] Furthermore, the hematological malignancy is acute lymphoblastic leukemia or acute myeloid leukemia.

[0014] Furthermore, the mass concentration of the GelMA solution is 5%-10%; the mass concentration of the photoinitiator is 0.01-0.05%.

[0015] As a preference, the mass concentration of the GelMA solution is 5%; the mass concentration of the photoinitiator is 0.05%.

[0016] Furthermore, in S02, 30,000-50,000 tumor cells are resuspended in each 25 μL of the bone marrow microenvironment-simulating matrix solution. The bone marrow microenvironment-simulating matrix provides an environment for the correct differentiation of the immune phenotype of the cultured hematological malignancy organoids.

[0017] Preferably, 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.

[0018] 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.

[0019] Furthermore, the immunophenotypic identification includes co-incubating the cultured hematological malignancy organoids with hematological malignancy-specific antibodies, detecting the expression of the specific antibodies on the hematological malignancy organoids; and using the expression of the specific antibodies on the patient's bone marrow cells as a comparison.

[0020] Preferably, the expression level of the specific antibody in the immunophenotyping identification on the hematological malignancy organoids differs by no more than 10% compared with that in the patient's bone marrow cells.

[0021] More preferably, the specific antibody is CD34.

[0022] More preferably, the first cytokine composition is specifically composed 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.

[0023] Another aspect of the present invention is to provide an application of the cultured blood tumor organoids.

[0024] The application of blood malignancy organoids cultured by the above-mentioned culture method in the preparation of preclinical drug resistance detection models.

[0025] Beneficial technical effects:

[0026] The present invention first proposes a method for standardized construction of organoids for hematological malignancies. According to the method provided by the present invention, multiple AML\ALL organoids have been cultured, proving that the method of the present invention has good reproducibility.

[0027] Secondly, the present invention utilizes non-epithelial bone marrow cells for the cultivation of hematologic tumor organoids, achieving rapid growth within just seven days of culture. This presents an advantage over existing solid tumor organoids, which typically utilize epithelial cells and undergo longer culture cycles. Furthermore, reports of successful hematologic tumor organoid cultivation are rare, thus demonstrating the significant novelty of the present invention's technical solution.

[0028] Furthermore, the method system provided by the present invention not only successfully cultured blood tumor organoids, but also further performed phenotypic identification thereof. The screened blood tumor organoids were similar to the patient's cell immune phenotype and can subsequently be used to prepare a drug resistance detection model. The drug resistance detection model can more accurately screen out which drugs clinically resistant patients are more sensitive to, thereby increasing the effect of disease treatment. Experimental verification has shown that the reason why the blood tumor organoids prepared by the present invention can more accurately reflect drug resistance is closely related to the promotion of the correct differentiation of the organoid immune phenotype in the preparation method.

[0029] The drug screening or drug sensitivity test results of the blood tumor organoids that are cultured and close to the patient's cell immune phenotype can provide more accurate data support for the patient's personalized precision treatment.

[0030] Finally, the culture method provided by the present invention features an innovative set of culture medium components and a bone marrow microenvironment-simulating matrix component. The culture medium utilizes a streamlined, yet high-concentration, first cytokine combination (specifically stem cell factor, FMS-like tyrosine kinase 3, interleukin-3, fibroblast growth factor 2, and bone morphogenetic protein 4) supplemented with high concentrations of specific cytokines. This allows for efficient culture of hematologic tumor organoids with high survival rates. Furthermore, the use of this innovative bone marrow microenvironment-simulating matrix component enables more accurate phenotypic differentiation of organoids compared to Matrigel, a commonly used matrix in the field. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the following is a brief introduction to the drawings required for the embodiments or the description of the prior art. In all drawings, similar elements or parts are generally identified by similar reference numerals. In the drawings, the various elements or parts are not necessarily drawn according to the actual scale. Obviously, the drawings described below are some embodiments of the present invention. For those of ordinary skill in the art, other drawings can also be obtained based on these drawings without inventive work.

[0032] Figure 1 This is a bright field image of the growth of acute lymphoblastic leukemia organoids in one of the embodiments of the present invention (scale bar 50 μm);

[0033] Figure 2 The results of a sensitivity test of ALL organoids cultured in one embodiment of the present invention to different anti-tumor drugs are shown;

[0034] Figure 3 This is a bright field image of the growth of acute myeloid leukemia under different culture conditions in one embodiment of the present invention;

[0035] Figure 4 The results of the drug sensitivity experiment of different AML organoids to venetoclax in one embodiment of the present invention are shown;

[0036] Figure 5 The results of drug sensitivity experiments of AML organoids in different culture systems in one embodiment of the present invention are as follows;

[0037] Figure 6 These are the results of a sensitivity test of AML organoids cultured in one embodiment of the present invention to different anti-tumor drugs. DETAILED DESCRIPTION

[0038] To make the purpose, technical solutions, and advantages of the embodiments of the present invention more clear, the technical solutions in the embodiments of the present invention will be clearly and completely described below in conjunction with the accompanying drawings in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without making creative efforts are within the scope of protection of the present invention.

[0039] As used herein, "and / or" includes any and all combinations of one or more of the associated listed items.

[0040] Herein, "plurality" means two or more than two, ie, it includes two, three, four, five, etc.

[0041] 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.

[0042] In this specification, certain embodiments may be disclosed in a format that is within a range. It should be understood that this description of "within a range" is merely for convenience and brevity and should not be interpreted as a rigid limitation on the disclosed range. Therefore, the description of a range should be considered to have specifically disclosed all possible subranges and individual numerical values ​​within this range. For example, the description of a range of 1-6 should be considered to have 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., as well as individual numbers within this range, such as 1, 2, 3, 4, 5, and 6. Regardless of the breadth of the range, the above rules apply.

[0043] Definition of noun:

[0044] The term "close to the patient's immune phenotype" as used in the present invention means that the results of flow cytometry identification show that the immune phenotype (surface markers) are similar to those of the patient's tumor cells.

[0045] Example 1

[0046] This example provides an example of a method for culturing, identifying, and drug screening organoids for hematological malignancies.

[0047] 1. Culture methods for hematological tumor organoids.

[0048] S01: Obtain a bone marrow or peripheral blood sample from the patient and obtain a tumor cell sample by density gradient centrifugation. Resuspend the tumor cell sample in an appropriate amount of red blood cell lysis buffer and lyse on ice for 3 minutes. Terminate lysis by adding DPBS. Pre-chill the centrifuge and centrifuge at 600g at 4°C for 5 minutes. Discard the supernatant. Resuspend the cells in an appropriate amount of a bone marrow microenvironment-mimicking matrix consisting of a 5% GelMA solution (containing 0.05% photoinitiator LAP). Plate the cells in a 48-well plate and allow to solidify.

[0049] S02: Hematologic tumor organoids are cultured in 200 μL of SFEM II medium (StemSpan SFEM II serum-free hematopoietic stem cell medium) supplemented with cytokines, including 1% 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). The organoid medium is changed every 2-3 days, and primary organoids are passaged after 7-10 days of culture. The resulting organoids are designated hematologic malignancy organoids, or hematologic tumor organoids.

[0050] 2. Characterization of Hematologic Tumor Organoids.

[0051] Flow cytometry was used to analyze the cell composition of organoids and detect the expression of antibodies such as CD45 and CD34.

[0052] 3. Drug Screening

[0053] Successfully cultured hematologic tumor organoids were digested with digestive fluid, washed with DPBS buffer, and centrifuged at 600g at 4°C for 5 minutes. The supernatant was discarded. The collected cells were counted, resuspended in culture medium, and plated at 1000 organoids per well in a 96-well plate. The test drug was added, and cell viability was tested 5 days later.

[0054] Example 2

[0055] This example provides an example of constructing acute lymphoblastic leukemia (ALL) organoids.

[0056] 1. Primary culture:

[0057] (1) The patient signed an informed consent form, and a bone marrow sample was taken from the patient. The sample was placed at room temperature for a period of time, rinsed with a pipette, and then diluted with room temperature PBS.

[0058] (2) First add 15 mL of sample density gradient separation solution into a 50 mL centrifuge tube, and then slowly add the diluted blood to the surface of the separation solution.

[0059] (3) Centrifuge at 400 g for 30 minutes at room temperature.

[0060] (4) Take the centrifuge tube out of the centrifuge. The liquid surface of the centrifuge tube is divided into four layers, namely, the first plasma layer, the second mononuclear cell layer, the third separation liquid layer, and the fourth red blood cell layer from top to bottom. Discard the top 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.

[0061] (5) After centrifugation, remove 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.

[0062] (6) After cell counting, take an appropriate number of cells, resuspend them in 5% GelMA (containing 0.05% photoinitiator LAP) and inoculate them into a 48-well plate (25 μL 5% GelMA resuspends 50,000 cells), solidify them by UV irradiation for 60 seconds, add ALL organoid-specific culture medium and culture them. Change the culture medium every 2-3 days. Passage every 7-10 days. The results of 7-day culture are shown in Figure 1 , the results showed that ALL organoids grew faster within 7 days.

[0063] 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).

[0064] As a control, after counting the cells, take an appropriate number of cells, resuspend them in Matrigel, and seed them into a 48-well plate (50,000 cells per 25 μL Matrigel). After solidification in an incubator, add ALL organoid-specific culture medium and culture. Change the medium every 2-3 days. Passage every 7-10 days.

[0065] 2. Subculture:

[0066] (1) After the organoids have grown to an appropriate number, they are digested and expanded. The culture medium in the cell culture plate is aspirated, and the organoid-GelMA mixed gel droplets are scraped with a 1 mL pipette tip and collected in a 15 mL centrifuge tube. The suspension is mixed by pipetting and centrifuged at 300 g for 5 min.

[0067] (2) Discard the supernatant and add 2-3 mL of GelMA digestion solution (EFL) according to the volume of GelMA after centrifugation, and blow gently with the pipette tip 4-5 times.

[0068] (3) Place the sample in a 37°C water bath for digestion. After complete digestion is observed under a microscope, terminate the digestion and centrifuge at 300 g for 5 minutes.

[0069] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5 minutes.

[0070] (5) Depending on the number of cell clusters, resuspend the cells in 5% GelMA and inoculate them into 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. Passage every 7-10 days.

[0071] 3. Cryopreservation:

[0072] (1) After the organoids have grown to an appropriate number, they are frozen. The culture medium in the cell culture plate is aspirated, PBS is aspirated, and the organoid-GelMA mixed gel droplets are scraped using a 1 mL pipette tip. The mixed suspension is collected in a 15 mL centrifuge tube. The suspension is mixed by pipetting with a pipette tip and centrifuged at 300 g for 5 min.

[0073] (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 a pipette tip that has been rinsed with the rinse solution.

[0074] (3) Place the sample in a 37°C water bath for digestion. After complete digestion is observed under a microscope, terminate the digestion and centrifuge at 300 g for 5 minutes.

[0075] (4) Discard the supernatant, resuspend the pellet with PBS, mix the cell suspension thoroughly, and centrifuge at 300g for 5 minutes.

[0076] (5) Freeze in liquid nitrogen.

[0077] 4. Resuscitation:

[0078] (1) Remove the cryovial from liquid nitrogen and thaw it in a 37°C water bath.

[0079] (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 minutes.

[0080] (3) Discard the supernatant, transfer the cell pellet to a 15 mL centrifuge tube with PBS, and centrifuge at 600 g for 5 min.

[0081] (4) Discard the supernatant and resuspend the cells in 5% GelMA according to the number of cell clusters. Inoculate the cells in a 48-well plate, irradiate with UV light for 30 seconds to solidify, add ALL organoid-specific culture medium, and replace the medium every 2-3 days. Passage every 7-10 days.

[0082] 5. Identification:

[0083] Flow cytometry: Single cells digested from bone marrow samples and ALL organoids cultured in the aforementioned steps were incubated with a combination of selected antibodies (CD34, HLA-DR, CD33, CD3, CD7, and CD13) to allow the antibodies to bind specifically to cell surface antigens. The flow cytometer was then turned on, preheated, and calibrated. The labeled samples were then placed in the flow cytometer for analysis. The results of the analysis are shown in Table 1.

[0084] Diagnostic criteria for ALL: ALL is diagnosed when the proportion of blasts (total nucleated cells) in the bone marrow is ≥ 20%. In this example, this refers specifically to the blast cell marker CD34. It should be noted that ALL can be divided into different subtypes, but this example does not identify ALL subtypes; it only determines whether the cultured organoids are ALL.

[0085] Table 1 Immune phenotype analysis and identification of ALL organoids

[0086]

[0087] The results showed that the binding of ALL organoids cultured in 5% GelMA to antibodies was almost identical to that of ALL patient bone marrow cells, indicating that the phenotype of the cultured ALL organoids was very close to that of real patients. In contrast, the binding of ALL organoids cultured in Matrigel matrix gel to antibodies differed significantly from that of ALL patient bone marrow cells, indicating that the phenotype of ALL organoids cultured in this method was far from that of real patients.

[0088] 6.Drug screening:

[0089] (1) When the ALL organoids have grown to sufficient size, collect the organoids, digest them into single cells, and count them.

[0090] (2) After centrifugation, discard the supernatant and ensure that the plating density is 500-1000 organoids per well based on the counting results.

[0091] (3) Prepare an appropriate amount of ALL organoid-specific culture medium in advance.

[0092] (4) Use a 1000 μL pipette to mix the cell suspension after adjusting the 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.

[0093] (5) For the blank group, only 100 μL of culture medium without cells was added to each well.

[0094] (6) After the plate is laid, PBS can be added to the surrounding wells to maintain humidity; the 96-well plate is placed in the incubator and waits for drug addition.

[0095] (7) Use a 200 μL pipette to add the prepared target concentrations of venetoclax: 10, 20, 40, 100 (μM); vindesine sulfate: 0.001, 0.01, 0.1, 1, 10 (nM) to the corresponding dosing groups in sequence, 100 μL of the drug solution per well, and add the drug along the side wall of the well with the tip of the pipette. Be sure to blow several times before adding to mix the drug solution.

[0096] (8) After aspirating the drug solution of different concentrations, replace the gun tip and proceed to the next step.

[0097] (9) Only 100 μL of culture medium was added to the negative group and blank group.

[0098] (10) After adding the drug, gently tap the culture plate to mix the drug solution and cells for full reaction.

[0099] (11) Place the plate in an incubator and perform ATP testing 5 days after drug exposure.

[0100] (12) The above-mentioned cultured ALL organoids were derived from the bone marrow cells of patients with clinical resistance to venetoclax. The results of drug testing of cultured ALL organoids are shown in Figure 2 The results showed that the organoid model was more sensitive to vindesine sulfate than venetoclax and showed resistance to venetoclax, which was consistent with the patient's drug response.

[0101] Example 3

[0102] This example provides an example of culturing and drug screening of acute myeloid leukemia (AML) organoids.

[0103] 1. Cultivation of Acute Myeloid Leukemia (AML) Organoids: The method was the same as in Example 2, and the AML organoid-specific culture medium used was as follows.

[0104] AML 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 25 ng / ml TPO (thrombopoietin).

[0105] AML organoid control medium: SFEM II medium (StemSpan SFEM II Hematopoietic Stem Cell Serum-Free 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), and 10 ng / ml BMP4 (bone morphogenetic protein 4).

[0106] 2. Test under different culture conditions: AML organoids were cultured in a similar manner as in Example 2, with one portion of the AML organoids cultured in AML organoid control medium and another portion of the AML organoids cultured in AML organoid-specific medium. Images were collected after 3 days of culture. Figure 3 As can be seen in the figure, the organoids in the TPO-added group grew faster and larger in size.

[0107] On the other hand, a portion of AML organoids were resuspended in Matrigel and supplemented with AML organoid-specific medium and cultured for 3 days; a portion of AML organoids were resuspended in 5% GelMA and seeded in a 48-well plate for light curing and supplemented with AML organoid-specific medium and cultured for 3 days. Figure 3 As can be seen in the figure, the organoid spheroids in 5% GelMA are larger in size and more in number than those in Matrigel.

[0108] The cultured AML organoids were immunophenotypically identified, and organoids with immunophenotypes close to those of the same patient's bone marrow cells were selected for subsequent experiments.

[0109] 3. Digestion of Organoids:

[0110] (1) When the organoids have grown to a sufficient amount, collect them, digest them into single cells, and count them.

[0111] (2) After centrifugation, discard the supernatant and ensure that the plating density is 500-1000 organoids per well based on the counting results.

[0112] 4. Cell plating:

[0113] (1) Prepare an appropriate amount of AML organoid-specific culture medium in advance.

[0114] (2) Use a 1000 μL pipette to mix the cell suspension after adjusting the 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.

[0115] (3) In the blank group, only 100 μL of culture medium without cells was added to each well.

[0116] (4) After the plate is plated, PBS can be added to the surrounding wells to maintain humidity. The 96-well plate is placed in an incubator and awaits drug addition. The drugs used in this example are venetoclax, azacitidine, decitabine, and gilteritinib.

[0117] 5. Drug effects

[0118] (1) Using a 200 μL pipette, add the prepared target concentrations of venetoclax: 50, 5, 0.5, 0.05, 0.005, 0.0005, 0.00005 (μM); azacitidine, decitabine, gilteritinib: 100, 10, 1, 0.1, 0.01, 0.001, 0.0001 (μM) to the corresponding drug-dosing groups in sequence, 100 μL of the drug solution per well, and add the drug along the side wall of the well with the tip of the pipette. Be sure to pipette several times before adding to mix the drug solution.

[0119] (2) After aspirating the drug solution of different concentrations, replace the gun tip and proceed to the next step.

[0120] (3) Only 100 μL of culture medium was added to the negative group and blank group.

[0121] (4) After adding the drug, gently tap the culture plate to mix the drug solution and cells for full reaction.

[0122] (5) Place the plate in an incubator and test the drug 5 days later.

[0123] 6. Testing:

[0124] After 5 days, ATP was detected.

[0125] 7. Data Analysis:

[0126] According to the ATP test results, data analysis was performed using GraphPad Prism. The experimental results are shown in the figure below. Figure 4As shown, AML organoid model 1 is derived from the bone marrow cells of a drug-naive patient, while AML organoid model 2 is derived from the bone marrow cells of a patient clinically resistant to venetoclax. In vitro drug sensitivity testing results showed that the model successfully retained the drug resistance characteristics of the original tumor. Notably, different organoids exhibited significant heterogeneity in response to the same drug, while traditional cell line models (MOLM-13 and HL60) showed high homogeneity in drug sensitivity responses, demonstrating that organoids have a greater advantage than traditional cell lines in simulating individual clinical differences.

[0127] Furthermore, to verify the effect of microenvironment matrix on drug resistance phenotype, the experiment further used two culture systems, matrigel and 5% GelMA hydrogel, for comparative study ( Figure 5 The experiments showed that: 1) In the experimental group with added venetoclax, AML organoid 2 cultured in Matrigel was sensitive to venetoclax, indicating a reversal of the drug-resistant phenotype, indicating that the organoid phenotype shifted during the Matrigel culture process (consistent with the results of ALL culture in Example 2). AML organoid 2 cultured in 5% GelMA hydrogel was resistant to venetoclax, consistent with the patient's drug-resistant phenotype. 2) In the Gilteritinib-treated group, AML organoid 2 cultured in 5% GelMA hydrogel was significantly more sensitive to gilteritinib, demonstrating that organoid models with a phenotype close to that of patients can more accurately screen for appropriate therapeutic drugs for drug-resistant patients.

[0128] further, Figure 6 The study demonstrated sensitivity testing of multiple drugs using organoid models derived from drug-naive patients. The results showed that the organoids exhibited distinct responses to different drugs, demonstrating varying sensitivities to different anti-tumor drugs. Because previous experiments have demonstrated that hematologic tumor organoid models cultured using the present method closely resemble patient phenotypes, the results from using these hematologic tumor organoids for preclinical drug screening are highly relevant.

[0129] It should be noted that, in this document, the terms "comprises," "includes," or any other variations thereof are intended to encompass non-exclusive inclusion, such that a process, method, article, or apparatus comprising a series of elements includes not only those elements but also other elements not explicitly listed, or elements inherent to such process, method, article, or apparatus. In the absence of further limitations, an element defined by the phrase "comprising a ..." does not exclude the presence of other identical elements in the process, method, article, or apparatus comprising the element.

[0130] The embodiments of the present invention are described above in conjunction with the accompanying drawings, but the present invention is not limited to the above-mentioned specific implementation methods. The above-mentioned specific implementation methods are merely illustrative and not restrictive. Under the guidance of the present invention, ordinary technicians in this field can also make many forms without departing from the scope of protection of the present invention and the claims, all of which are protected by the present invention.

Claims

1. A method for culturing organoids of hematological malignancies with an immunophenotype close to that of a patient, characterized in that: The following steps are involved: S01: Obtain a cell sample from a patient with a hematological malignancy, wherein the cell sample is a tumor cell sample derived from bone marrow or a tumor cell sample derived from peripheral blood; the tumor cell sample is an acute lymphoblastic leukemia cell sample or an acute myeloid leukemia cell sample; S02: preparing a bone marrow microenvironment simulation matrix, wherein the bone marrow microenvironment simulation matrix is ​​a methacrylated gelatin solution containing a photoinitiator, and resuspending the tumor cell sample in the bone marrow microenvironment simulation matrix solution for UV curing; S03: adding a specific culture medium to the bone marrow microenvironment-simulating matrix of S02 to culture hematological malignancy organoids, wherein the specific culture medium comprises a basal culture medium and cytokines; the basal culture medium is a serum-free culture medium for hematopoietic stem cells; and the cytokines comprise a first cytokine composition and specific cytokines; 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; When the cultured blood malignancy cell sample is an acute lymphoblastic leukemia cell sample, the specific cytokine added to the culture medium is interleukin-7; When the cultured blood malignancy cell sample is an acute myeloid leukemia cell sample, the specific cytokine added to the culture medium is thrombopoietin; S04: Identify the immunophenotype of the cultured hematological malignancy organoids; screen out hematological malignancy organoids that have an immunophenotype close to that of the patient's bone marrow cells.

2. The culture method according to 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 culture method according to claim 1, wherein In S02, 30,000-50,000 tumor cells were resuspended in each 25 μL of the bone marrow microenvironment-simulating matrix solution.

4. The culture method 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.

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

6. The culture method according to claim 5, wherein The expression level of the specific antibody in the immunophenotyping assay on the hematological malignancy organoids is no more than 10% different from that on the patient's bone marrow cells.

7. The culture method according to claim 4, 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.

8. Use of hematological malignancy organoids cultured by the culture method according to any one of claims 1 to 7 in preparing a preclinical drug resistance detection model.

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