Culture medium and culture method for human primary acute myeloid leukemia cells

Abstract

A culture medium and a culture method for human primary acute myeloid leukemia cells are provided. The culture medium for human primary acute myeloid leukemia cells includes glutamine additive, non-essential amino acids, human interleukin-6, human interleukin-7, human interleukin-3, recombinant human FLT3 ligand, recombinant human macrophage colony-stimulating factor, and human stem cell factor. By using the above culture medium and culture method, acute myeloid leukemia cells can be cultured with higher expansion efficiency and longer in-vitro culture time. Also provided are human primary acute myeloid leukemia cells cultured in vitro using the culture medium, and its use for evaluating and screening the therapeutic effect of drugs.

Description

[Technical Field]

[0001] The present invention relates to the field of cell culture technology, in particular to a primary cell culture medium for culturing human primary acute myeloid leukemia (AML) cells in vitro, a method for culturing human primary acute myeloid leukemia cells using the culture medium, and their application in drug efficacy evaluation and screening. [Background technology]

[0002] Acute myeloid leukemia (AML) is a clonal malignant proliferative blood disorder of myeloblasts in the hematopoietic system. AML is a progressive, highly allogenic disease with biologically and prognostically distinct subtypes. AML affects 1 to 5 people per 100,000 each year, accounting for 30% to 40% of all new leukemia cases. AML is the most lethal leukemia disorder and has the worst prognosis and survival rate (less than 26% at 5 years), which has not improved since the 1970s (Non-Patent Document 1).

[0003] AML stem cells have been the primary focus of leukemia research to date, as they mediate resistance and relapsed leukemic subclones. Research work typically requires the culture of AML stem cells. Culturing AML stem cells is challenging due to heterogeneity within the AML stem cell population and the fact that many patients have poorly growing cells that tend to differentiate and lose their self-renewal capacity in vitro. Furthermore, primary AML patient samples can be difficult to obtain, and unlike transformed cell lines, these cannot be expanded indefinitely in vitro. In vivo xenograft models allow for the expansion of human AML cells, but require the cultivation of large numbers of cells (10 per mouse). 6 However, co-culture with bone marrow (BM)-derived mesenchymal stromal cells (MSCs) or stromal cell lines can help preserve AML stem cells, but feeder cell cultures are not suitable for high-throughput drug screening or adaptive testing.

[0004] Therefore, there is a need in the art for a culture medium and method that allows for the rapid long-term expansion of primary human acute myeloid leukemia cells without co-culture with stromal cells. [Prior art documents] [Non-patent literature]

[0005] [Non-Patent Document 1] Hanyang Lin, et al., "Feeder-free and serum-free in vitro assay for measuring the effect of drugs on acute and chronic myeloid leukemia stem / progenitor cells," Experimental Hematology 2020; 90: 52-64 Summary of the Invention

[0006] To solve the above technical problems, the present invention provides a culture medium and a culture method for rapidly expanding primary human acute myeloid leukemia cells in vitro.

[0007] One aspect of the present invention is to provide a culture medium for human primary acute myeloid leukemia cells, the culture medium comprising a glutamine supplement, non-essential amino acid(s), human interleukin-6 (human IL-6), human interleukin-7 (human IL-7), human interleukin-3 (human IL-3), recombinant human FLT3 ligand (human FLT3L), recombinant human macrophage colony-stimulating factor (human M-CSF), and human stem cell factor (human SCF).

[0008] In a preferred embodiment of the present invention, the culture medium for human primary acute myeloid leukemia cells is prepared under the following conditions: (1) The amount of glutamine additive in the culture medium is preferably 0.5 mM to 4 mM; (2) the non-essential amino acid is one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the amount of the non-essential amino acid in the culture medium is preferably 12.5 μM to 200 μM; (3) the amount of human IL-6 in the culture medium is preferably 1.89 ng / mL to 17 ng / mL; (4) the amount of human IL-7 in the culture medium is preferably 1.89 ng / mL to 51 ng / mL; (5) The amount of human IL-3 in the culture medium is preferably 1.89 ng / mL to 153 ng / mL; (6) The amount of human FLT3L in the culture medium is preferably 3 ng / mL to 81 ng / mL; (7) The amount of human M-CSF in the culture medium is preferably 1 ng / mL to 81 ng / mL; (8) The amount of human SCF in the culture medium is preferably 1 ng / mL to 81 ng / mL; Satisfy one or more or all of the following.

[0009] In another preferred embodiment, the culture medium for human primary acute myeloid leukemia cells of the present invention further comprises an initial medium selected from the group consisting of monocyte serum-free medium and RPMI-1640, and a basal medium containing 5% to 10% (volume / volume) fetal bovine serum and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B, and primocin. In detail, when streptomycin / penicillin is used, the concentration range of streptomycin is 25 μg / mL to 400 μg / mL, preferably 50 μg / mL to 200 μg / mL, and the concentration range of penicillin is 25 U / mL to 400 U / mL, preferably 50 U / mL to 200 U / mL. When amphotericin B is used, the concentration range is 0.25 μg / mL to 4 μg / mL, preferably 0.5 μg / mL to 2 μg / mL. When primocin is used, the concentration range is 25 μg / mL to 400 μg / mL, preferably 50 μg / mL to 200 μg / mL.

[0010] On the other hand, the present invention also provides a method for culturing human primary acute myeloid leukemia cells in vitro, comprising the step of culturing human primary acute myeloid leukemia cells in vitro using the culture medium for human primary acute myeloid leukemia cells of the present invention.

[0011] In a preferred embodiment, the method of culturing human primary acute myeloid leukemia cells in vitro of the present invention comprises the following steps:

[0012] 1. Isolating and processing human primary acute myeloid leukemia cells (1) Centrifuge a bone marrow sample from a patient with acute myeloid leukemia at 1200 rpm to 1600 rpm for 2 to 6 minutes. (2) After centrifugation, discard the upper plasma layer, add 1x PBS in an amount 2 to 3 times the volume of the hemocyte sediment, dilute the hemocyte sediment, mix thoroughly, add 6 mL to 8 mL of human peripheral blood lymphocyte separation medium to the diluted hemocyte sediment, and centrifuge the resultant at a speed of 380 g to 420 g, an acceleration of 1 to 2, a deceleration of 0, a temperature of 20°C to 28°C, and a centrifugation time of 25 to 35 minutes. (3) After centrifugation to form layers in the centrifuge tube, aspirate the lymphocyte layer into 3 mL to 6 mL of 1x PBS, mix well to wash the cells, and then centrifuge at 1200 rpm to 1600 rpm for 2 to 6 minutes. (4) Discard the supernatant, add red blood cell lysis buffer to resuspend the cell pellet, lyse for 15 to 20 minutes, and then centrifuge at 1200 rpm to 1600 rpm for 2 to 6 minutes. (5) After centrifugation, discard the supernatant and add basal medium for later use.

[0013] 2. Culturing cells using the culture medium for human primary acute myeloid leukemia cells of the present invention The human primary acute myeloid leukemia cells obtained in step 1 above were resuspended in the culture medium for human primary acute myeloid leukemia cells of the present invention, the cells were counted, and the cells were separated into 1 cm 2 1x10 5 pieces~4×10 6 The cells are seeded into culture dishes at a density of 1000 cells / mL and passaged when they grow to cover 90% of the culture dish.

[0014] In yet another aspect, the cells obtained by the culture method of the present invention can be used in regenerative medicine, basic medical research on acute myeloid leukemia cells, screening of drug response, and development of new drugs for acute myeloid leukemia. Thus, the present invention also provides a method for screening or evaluating the efficacy of drugs for human primary acute myeloid leukemia, comprising: (1) culturing human primary acute myeloid leukemia cells by the method for culturing human primary acute myeloid leukemia cells of the present invention; (2) selecting a drug to be tested and diluting the drug into various concentration gradients; (3) adding the diluted drug to the cells obtained by culturing in step (1) and detecting the cell viability; The present invention provides a method comprising:

[0015] The technical solution of the present invention can achieve the following technical effects: (1) The success rate of culturing primary human acute myeloid leukemia cells has improved, reaching over 80%. (2) Primary human acute myeloid leukemia cells cultured in vitro reliably maintain the pathological characteristics of patients. (3) Human primary acute myeloid leukemia cells were expanded with high efficiency and maintained for 10 5 Within approximately one week of the start of cell counts at the 10 6 Human primary acute myeloid leukemia cells were successfully expanded to a scale of 100 cells, and the expanded human primary acute myeloid leukemia cells have the ability to be serially passaged. (5) The culture medium does not require expensive factors such as Wnt agonists, R-spondin family proteins, BMP inhibitors, and FGF10, which reduces culture costs. (6) This technology can culture and provide large quantities of human primary acute myeloid leukemia cells with high uniformity, which are suitable for high-throughput screening of new candidate compounds and high-throughput in vitro drug sensitivity functional testing in patients. [Brief explanation of the drawings]

[0016] [Figure 1] 1 is a graph showing the effect of various combinations of added factors on the proliferation of human primary acute myeloid leukemia cells. [Figure 2-1] 1 is a graph showing the effect of various concentrations of each added factor on the proliferation of human primary acute myeloid leukemia cells. [Figure 2-2] 1 is a graph showing the effect of various concentrations of each added factor on the proliferation of human primary acute myeloid leukemia cells. [Figure 3] 1 is a photograph taken under a microscope of human primary acute myeloid leukemia cells cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention. [Figure 4] 1 is an image showing specific results of flow cytometry of human primary acute myeloid leukemia cells cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention. [Figure 5] 1 shows a cell growth curve of human primary acute myeloid leukemia cells cultured in vitro using the culture medium for human primary acute myeloid leukemia cells of the present invention. [Figure 6] 1 is a graph comparing the culture of human primary acute myeloid leukemia cells using a culture medium for human primary acute myeloid leukemia cells of the present invention and a culture medium of the prior art. [Figure 7] 1 shows dose-response curves for various drugs of various passages of human primary acute myeloid leukemia cells cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention. DETAILED DESCRIPTION OF THE INVENTION

[0017] For a better understanding of the present invention, the present invention will be further described below in combination with examples and figures. The following examples are intended to be merely illustrative of the present invention and are not intended to be a definition thereof.

[0018] Example 1. Effects of each added factor in the culture medium for human primary acute myeloid leukemia cells on the proliferation of human primary acute myeloid leukemia cells (1) Preparation of culture medium for human primary acute myeloid leukemia cells First, a basal medium was prepared, which consisted of Monocyte Serum-Free Medium (purchased from BI, 05-080-1A) + 10% (v / v) fetal bovine serum (purchased from ExCell Bio, FND500) + 100 μg / mL primocin (purchased from InvivoGen, 0.2% (v / v); the commercially available product concentration is 50 mg / mL).

[0019] Various types of growth factors (see Table 1) were added to the basal medium to prepare culture media for human primary acute myeloid leukemia cells containing various added components.

[0020] (2) Isolation and processing of human primary acute myeloid leukemia cells 1. Sample Selection Bone marrow samples were obtained from AML patients by specialized medical staff at specialized medical institutions, and all patients signed informed consent forms. Bone marrow samples (3 mL to 10 mL) were stored in EDTA-K2 anticoagulant tubes (manufacturer: Jiangsu Rongye) and transported refrigerated at 4°C to 8°C.

[0021] 2. Preparation of Materials After surface sterilization, a 15 mL sterile centrifuge tube, a pipettor, a 10 mL pipette, and sterile pipette tips were placed in an ultra-clean workbench and exposed to UV light for 30 minutes. 1x PBS was removed from the 4°C refrigerator 30 minutes prior to the sterilization.

[0022] 3. Sample Separation 3.1 In an ultra-clean workbench, the bone marrow sample was mixed by pipetting up and down, transferred to a 15 mL centrifuge tube, and centrifuged at 1500 rpm for 4 minutes at room temperature.

[0023] 3.2 Add 6 mL of human peripheral blood lymphocyte separation medium (purchased from Solarbio, P8610) to a new 15 mL centrifuge tube. After centrifugation of the bone marrow sample, discard the upper plasma layer. Dilute the blood sediment with 1x PBS (2-3 times the volume of the blood sediment). Mix thoroughly and carefully to keep the liquid surface clear. Slowly place the diluted blood on the surface of the separation medium layer along the wall of the centrifuge tube. Gently place the centrifuge tube containing the bone marrow sample mixture in a centrifuge and centrifuge at 400 g for 30 minutes (acceleration: 2, deceleration: 0, temperature: 25°C).

[0024] 3.3 After centrifugation, the cells in the centrifuge tube were separated into four layers from top to bottom (PBS layer, ring-shaped milky white lymphocyte layer, separation medium layer, and red blood cell layer). The lymphocyte layer was transferred with a circular pipette into a 15 mL centrifuge tube to which 5 mL of 1×PBS had been added in advance. The resulting mixture was gently mixed to wash the cells, and centrifuged at 1500 rpm for 5 minutes at room temperature.

[0025] 3.4 The supernatant was discarded and the resultant was observed to determine whether blood cells were present. If blood cells were present, 8 mL of red blood cell lysis buffer (purchased from Sigma, R7757-100ML) was added, which was then mixed well and inverted once during the process to lyse the blood cells at 4°C for 20 minutes. The resultant was centrifuged at 1500 rpm for 4 minutes at room temperature.

[0026] 3.5 The supernatant was discarded and 2 mL of basal medium was added to the resultant to resuspend the cells for later use.

[0027] 4. Cell Counting and Processing 4.1 Viable cell counting: 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue dye (Sangon Biotech (Shanghai) Co., Ltd.), and then 20 μL of the mixture was added to a cell counting plate (Countstar, specification: 50 per box), and the percentage of viable large cells (cell size greater than 10 μm) was calculated using a cell counter (Countstar, IC1000) (percentage of viable large cells (cell size greater than 10 μm) = number of viable cells / total number of cells × 100%).

[0028] (3) Culture of primary human acute myeloid leukemia cells Culture media with various compositions shown in Table 1 were added to a 96-well plate at a volume of 100 μL per well. Following step (2) above, 1 × 10 primary human acute myeloid leukemia cells isolated from two primary human acute myeloid leukemia bone marrow samples (numbered A17007 and A25104 from the First Affiliated Hospital of Anhui Medical University) were added per well. 4Cells were seeded into 96-well culture plates at a density of 100 cells / well and cultured at 37°C and 5% CO2. After 5 to 8 days of culture, when the cells had grown to 70% to 85%, 10 μL of Cell Counting Kit-8 (CCK-8, purchased from MCE) was added to each well and incubated at 37°C and 5% CO2 for 2 to 4 hours. The materials in each well were mixed, and the plate was read at 450 nm using a multifunctional microplate reader (multimode detection platform, American Molecular Instruments (Shanghai) Co., Ltd.). Basal medium without any additives was used as a control. The experimental results are shown in Table 1.

[0029] [Table 1]

[0030] In the table, "+" indicates that, compared with the basal medium, the culture medium to which the additive has been added can promote the proliferation of two primary human acute myeloid leukemia cells isolated from primary human acute myeloid leukemia bone marrow samples; "-" indicates that the culture medium to which the additive has been added can promote the proliferation of at least one primary human acute myeloid leukemia cell isolated from primary human acute myeloid leukemia bone marrow samples; and "○" indicates that the culture medium to which the additive has been added has no significant effect on the proliferation of at least two primary human acute myeloid leukemia cells isolated from primary human acute myeloid leukemia bone marrow samples.

[0031] Based on the above results, factors such as human IL-7, human IFN-α, human M-CSF, human SCF, human IL-6, glutamine supplement, human FLT3L, human IL-3, and non-essential amino acids were selected for further culture experiments in Example 2.

[0032] Example 2. Effects of various combinations of added factors in the culture medium for human primary acute myeloid leukemia cells on the proliferation of human primary acute myeloid leukemia cells Culture media for human primary acute myeloid leukemia cells containing various combinations of added factors were prepared according to the ingredients in Table 2 to investigate the growth-promoting effects of various combinations of added factors on human primary acute myeloid leukemia cells.

[0033] [Table 2]

[0034] Human primary acute myeloid leukemia cells were obtained from human primary acute myeloid leukemia bone marrow samples (numbered A17151, A17152, A20090, A20101, A21004, and A20141) according to the process of step (2)-3 of Example 1. The obtained cell suspension was divided into 11 equal portions, which were then centrifuged at 1500 rpm for 4 minutes. After centrifugation, the cells were resuspended in 200 μL of BM or culture medium No. 1 to No. 10, respectively, and 2 × 10 cells were collected per well. 4 The cells were seeded into a 48-well plate at a viable cell density of 20,000 cells per well. Each well in the 48-well plate was supplemented with the corresponding culture medium up to a volume of 1 mL, and the resulting mixture was thoroughly mixed. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation.

[0035] The cells in the 48-well plate grew to over 85% and were transferred to a 15 mL centrifuge tube, which was then centrifuged at 1500 rpm for 5 minutes. 500 μL of monocyte serum-free medium was added to the centrifuge tube to resuspend the cell pellet. 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue dye (Sangon Biotech (Shanghai) Co., Ltd.), and 20 μL of the mixture was added to a cell counting plate (Countstar, specifications: 50 cells per box). The percentage of viable large cells (cell size greater than 10 μm) was calculated using a cell counter (Countstar, IC1000) (percentage of viable large cells (cell size greater than 10 μm) = number of viable cells / total number of cells × 100%). The results obtained from human primary acute myeloid leukemia cells from bone marrow samples A17151, A17152, A20090, A20101, A21004, and A20141 are shown in Figure 1 .

[0036] The results in Figure 1 show that, compared with the basal medium, the use of culture media Nos. 1 to 10 can promote the proliferation of human primary acute myeloid leukemia cells to varying degrees. Culture medium No. 2 does not contain human IFN-α, which surprisingly results in a better proliferation effect. These results indicate that factors such as glutamine supplement, human SCF, human IL-6, human IL-3, human FLT3L, non-essential amino acids, human IL-7, and human M-CSF can significantly promote the proliferation of human primary acute myeloid leukemia cells.

[0037] Example 3. Proliferative effects of various concentrations of added factors on human primary acute myeloid leukemia cells Human primary acute myeloid leukemia cells were obtained from human primary acute myeloid leukemia bone marrow samples (numbered A23065, A17112) according to the process in step (2)-3 of Example 1. These cells were resuspended in basal medium (monocyte serum-free medium + 10% (vol / vol) fetal bovine serum + 100 μg / mL primocin) for later use.

[0038] Next, the factors having the cell culture growth-promoting effect determined in Example 2 were added to the basal medium (prepared according to the formulation of No. 2 in Example 2) to obtain a combined basal medium, and then the following eight types of culture medium formulations were prepared for the experiments: Formulation 1: Combined basal medium without glutamine supplement; Formulation 2: Combined basal medium without human SCF, Formulation 3: Combined basal medium without human IL-6, Formulation 4: Combined basal medium without human IL-3, Formulation 5: Combined basal medium without human FLT3L, Formula 6: Combined basal medium without non-essential amino acids, Formulation 7: Combined basal medium without human IL-7, Formulation 8: Combined basal medium without human M-CSF.

[0039] 4×10 4 20 μl of cell suspension containing 100 cells was added to each well, and the suspension was diluted with 1 mL of the medium of each of the above formulations 1 to 8.

[0040] When using the culture medium of formulation 1, the prepared glutamine additive was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of the glutamine additive being 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM, respectively. A blank control (BC) well was also prepared using the culture medium of formulation 1.

[0041] When using the culture medium of formulation 2, the prepared human SCF was added to each well of a 48-well plate seeded with primary cells at 1 mL per well, with the final concentrations of human SCF being 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. A blank control (BC) well was prepared using the culture medium of formulation 2.

[0042] When using the culture medium of formulation 3, the prepared human IL-6 was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of human IL-6 being 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. Blank control (BC) wells were prepared using the culture medium of formulation 3.

[0043] When using the culture medium of formulation 4, the prepared human IL-3 was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of human IL-3 being 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. A blank control (BC) well was prepared using the culture medium of formulation 4.

[0044] When using the culture medium of formulation 5, the prepared human FLT3L was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of human FLT3L being 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. A blank control (BC) well was prepared using the culture medium of formulation 5.

[0045] When using the culture medium of Formulation 6, the prepared non-essential amino acids were added to a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of the non-essential amino acids being 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively. A blank control (BC) well was also prepared using the culture medium of Formulation 6.

[0046] When using the culture medium of formulation 7, the prepared IL-7 was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final IL-7 concentrations being 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. A blank control (BC) well was also prepared using the culture medium of formulation 7.

[0047] When using the culture medium of formulation 8, the prepared human M-CSF was added to each well of a 48-well plate inoculated with primary cells at 1 mL per well, with the final concentrations of human M-CSF being 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. A blank control (BC) well was also prepared using the culture medium of formulation 8.

[0048] When the cells were expanded to approximately 85% of the 48-well plate, the proliferation fold was calculated by comparing the number of cells in the blank control (BC) wells. The results are shown in Figures 2A-2H. In Figures 2A-2H, the ratios represent the ratio of the number of primary passage cells cultured using each culture medium to the number of primary passage cells cultured in the corresponding blank control well. A ratio greater than 1 indicates that the proliferation-promoting effect of the prepared culture medium containing various concentrations of factors or small molecule compounds is more favorable than that of the culture medium in the blank control wells. A ratio less than 1 indicates that the proliferation-promoting effect of the prepared culture medium containing various concentrations of factors or small molecule compounds is less favorable than that of the culture medium in the blank control wells.

[0049] According to the results in Figures 2A to 2H, the glutamine additive, human SCF, human IL-6, human IL-3, human FLT3L, non-essential amino acids, human IL-7, and human M-CSF have a significant proliferation-promoting effect on primary human acute myeloid leukemia cells. According to the results of this example, the amount of glutamine additive is preferably 0.5 mM to 4 mM, and the cell proliferation effect is most significant when added at a concentration of 0.5 mM. The amount of human SCF is preferably 1 ng / ml to 81 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 9 ng / ml. The amount of human IL-6 is preferably 1.89 ng / ml to 17 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 5.67 ng / ml. The amount of human IL-3 is preferably 1.89 ng / ml to 153 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 51 ng / ml. The cell proliferation effect is also significant, the amount of human FLT3L is preferably 3 ng / ml to 81 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 27 ng / ml; the amount of non-essential amino acids is preferably 12.5 μM to 200 μM, and the cell proliferation effect is most significant when added at a concentration of 50 μM; the amount of human IL-7 is preferably 1.89 ng / ml to 51 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 17 ng / ml; and the amount of human M-CSF is preferably 1 ng / ml to 81 ng / ml, and the cell proliferation effect is most significant when added at a concentration of 27 ng / ml.

[0050] Example 4. Cultivation and characterization of human primary acute myeloid leukemia cells (1) Culture of primary human acute myeloid leukemia cells Human primary acute myeloid leukemia cells were obtained from a human primary acute myeloid leukemia bone marrow sample (numbered A17030) according to the process of step (2)-3 of Example 1, and cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention (the composition is a combination of optimal components and concentrations determined in Example 3, i.e., containing a basal medium, 0.5 mM glutamine supplement, 9 ng / mL human SCF, 5.67 ng / mL human IL-6, 51 ng / mL human IL-3, 27 ng / mL human FLT3L, 50 μM non-essential amino acids, 17 ng / mL human IL-7, and 27 ng / mL human M-CSF). The obtained human primary acute myeloid leukemia cells were cultured at a density of 3 × 10 per well. 6 The cells were seeded into a 6-well plate at a viable cell density of 1000 cells / well. Five milliliters of the culture medium for human primary acute myeloid leukemia cells of the present invention was added to the plate, and the resulting mixture was mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation.

[0051] Cultured human primary acute myeloid leukemia cells were observed under a microscope (EVOS M500, Invitrogen). Figure 3 shows photographs of cultures taken under a 10x objective lens after 1, 4, and 7 days of culture. Cell counting revealed a 3.58-fold increase in the number of viable cells after 7 days of culture.

[0052] (2) Flow cytometric identification of primary human acute myeloid leukemia cells Human primary acute myeloid leukemia cells were obtained from a human primary acute myeloid leukemia bone marrow sample (numbered A17014) according to the process of step (2)-3 of Example 1, and cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention. Specifically, the obtained human primary acute myeloid leukemia cells were cultured at 1 × 10 per well. 6 The cells were seeded into a 12-well plate at a viable cell density of 1000 cells / well. 3 mL of the culture medium of the present invention was added to the plate, and the resulting mixture was mixed thoroughly. After surface disinfection, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C and 5% CO2 for incubation.

[0053] Human primary acute myeloid leukemia cells before and after 7 days of culture were transferred to 15 mL centrifuge tubes and centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was diluted with 2 mL of 1x PBS. The cells were then divided into two equal portions in 1.5 mL centrifuge tubes. One portion was used for the experimental group double-labeled with a leukocyte marker (APC mouse anti-human CD45 (purchased from BD, 560973)) and a myeloid marker (BB515 mouse anti-human CD33 (purchased from BD, 564588)), and the other portion was used as a control group. These portions were then centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and 40 μL of 0.5% BSA (prepared in 1x PBS) was added to the centrifuge tube. The above antibodies were then added to the cells at a 1:40 ratio in the dark and mixed thoroughly. No antibodies were added to the control group. The cells were incubated on ice for 1 to 2 hours. After incubation, each tube was resuspended and washed with 1 mL of 1x PBS and centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, and the cell pellet was resuspended in 300 μL of 1x PBS. The expression of myeloid markers in the human primary acute myeloid leukemia cells was analyzed before and after 7 days of culture using flow cytometry (Beckman EVOS M500).

[0054] Figure 4 shows the results of flow cytometry of primary human acute myeloid leukemia cells cultured using the culture medium for primary human acute myeloid leukemia cells of the present invention. From Figure 4, it can be seen that the proportion of myeloid leukemia cells increased by 16% after 7 days of continuous culture of primary human acute myeloid leukemia cells cultured in the medium of the present invention.

[0055] Example 5. Statistics of primary culture period and cell number of human primary acute myeloid leukemia cells and calculation of population doubling (PD) value Following the process of step (2)-3 in Example 1, human primary acute myeloid leukemia cells were obtained from eight human primary acute myeloid leukemia cell bone marrow samples (numbered A23123, A15094, A23133, A23123-2, A23023, A14003, A23124, and A09169). The obtained human primary acute myeloid leukemia cells were cultured at 1 × 10 per well. 6 Cells were seeded into 12-well plates at a viable cell density of 1000 cells and cultured using the medium of the present invention. After 5 to 9 days of cell culture, the cells were passaged and counted, and the number of culture days up to the time of passage was considered as a culture cycle. Under these experimental conditions, the expanded cells were expanded at various passages. After each passage, the cells were counted and the corresponding culture cycle was recorded. The PD value was calculated according to the formula: population doubling (PD) = 3.32 × log10 (total number of cells after digestion / initial number of cells seeded). For this formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0056] Figure 5 shows the growth curves of eight primary cells cultured using the culture medium for human primary acute myeloid leukemia cells of the present invention, plotted using Graphpad Prism software. The horizontal axis represents the number of days in cell culture, and the vertical axis represents the cumulative cell expansion fold, i.e., the fold increase in cell expansion during a culture cycle. The larger this value, the higher the fold increase in cells within a given cycle, i.e., the more cells are expanded. The slope represents the rate of cell expansion. Figure 5 confirms that when human primary acute myeloid leukemia cells cultured in the culture medium of the present invention were continuously cultured and expanded for at least 45 days, the cell expansion rate remained essentially unchanged, and the cells still had the ability to continue expanding.

[0057] Example 6. Comparison of culture effects with conventional culture media (1) Preparation of control culture medium A conventional control culture medium (Silvia Ravera et al., Scientific Reports, (2020) 10:16519) was prepared by mixing 1640 culture medium (purchased from Corning, 10-040-CVR) + 10 ng / mL IL-15 (purchased from Sino Biological) + 10 ng / mL IL-4 (purchased from Sino Biological) + 10% FBS (purchased from excellbio, FND500) (hereinafter referred to as the "control culture medium").

[0058] (2) Obtaining and culturing primary human acute myeloid leukemia cells Human primary acute myeloid leukemia cells were obtained from a human primary acute myeloid leukemia bone marrow sample (A10093) according to the process of step (2)-3 of Example 1, and 1 × 10 cells were cultured per well. 6 The cells were seeded into a 12-well plate at a viable cell density of 1000 cells / well and cultured in the culture medium of the present invention and the control culture medium, respectively.

[0059] On day 7 of culture, the 12-well plate was removed. The cell culture was transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes at room temperature. The cell pellet was resuspended in 1 mL of culture medium. 12 μL of the resuspended cell suspension was thoroughly mixed with 12 μL of trypan blue dye (Sangon Biotech (Shanghai) Co., Ltd.), and 20 μL of the mixture was added to a cell counting plate (Countstar, specification: 50 cells per box). The total number of cells was counted using a cell counter (Countstar, IC1000). The counting results are shown in Figure 6.

[0060] The results in Figure 6 show that, compared with the control culture medium, the culture medium for human primary acute myeloid leukemia of the present invention can significantly promote the expansion and proliferation of human primary acute myeloid leukemia cells, and its effect is superior to that of the control culture medium.

[0061] Example 7. Drug screening and efficacy evaluation using human primary acute myeloid leukemia cells expanded using the culture medium of the present invention 1. Cell Culture and Plating Following the same process as in Example 1, human primary acute myeloid leukemia cells (numbered A23170) were isolated and used for one generation. Using the culture medium for human primary acute myeloid leukemia cells of the present invention, the cells were cultured until they were expanded to 85%, and then passaged. The cells were passaged and counted according to the steps in Example 1. The cells were cultured at 1 x 10 per mL. 5 The cells were placed in a loading slot (purchased from Corning) at a viable cell density of 1000 cells and mixed thoroughly. They were then placed in a 384-well opaque white cell culture plate (purchased from Corning) at a volume of 50 μL per well and a cell count of 5,000 cells per well. The plate was sealed by adding the culture medium for human primary acute myeloid leukemia cells of the present invention from the edge of the plate, and the sample name, dosing time, and test time using CellTiter-Glo (Promega) were marked on the plate. The surface of the plate was disinfected with 75% alcohol (LIRCON), and the plate was cultured in an incubator at 37°C and 5% CO2, followed by dosing after 24 hours. Cells from the first, second, third, fourth, and fifth passages of the culture were obtained for drug screening, and the drug sensitivity of the primary cells cultured using the culture medium of the present invention for serial passages was tested.

[0062] 2. Preparation of Candidate Drugs Six concentration gradients of six drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, and homoharringtonine, all purchased from MCE) were prepared according to the table below, and these were added to a 384-well plate (Thermo Fisher) at a volume of 30 μL per well and stored for use.

[0063] [Table 3]

[0064] 3. High-throughput Dosing The prepared drug plate was removed and kept at room temperature. The plate was centrifuged at 1000 rpm for 1 minute in a centrifuge (Beckman) at room temperature and then removed. A high-throughput automated workstation (JANUS, Perkin Elmer) was used for high-throughput dosing. 0.1 μL of the corresponding concentration of candidate drug was added to each well of a 384-well plate containing cultured human primary acute myeloid leukemia cells. After dosing, the surface of the 384-well plate was disinfected and transferred to an incubator. Cell viability was measured after 72 hours.

[0065] 4. Measuring Cell Viability CellTiter-Glo luminescent reagent (Promega) was removed from a 4°C refrigerator, and 10 mL of the reagent was added to the loading slot. The 384-well plate to be tested was removed from the incubator, and 10 μL of CellTiter-Glo luminescent reagent was added to each well. After allowing to stand for 10 minutes, the test was performed using a multifunction microplate reader (Envision, Perkin Elmer).

[0066] 5. Data Processing The cell inhibition rate of cells treated with various drugs was calculated according to the formula: Cell inhibition rate (%) = 100% - chemiluminescence value of drug-added wells / chemiluminescence value of control wells × 100%, and the median inhibitory rate (IC) of the drug on the cells was calculated. 50 ) was calculated using graphpad prism software. The results are shown in Figures 7A to 7F.

[0067] Figures 7A-7F show that when primary human acute myeloid leukemia cells cultured from the culture medium for primary human acute myeloid leukemia cells of the present invention are used for drug screening, the inhibitory effect of the same drug on cultured cells of various passages remains substantially the same (the inhibition curves are substantially consistent). Cells from the same patient have different sensitivities to different drugs at the maximum blood concentration in the human body. These results allow us to determine the effectiveness of this drug in clinical use in patients with primary human acute myeloid leukemia. At the same time, these results demonstrate that the drug sensitivity of tumor cells of various passages obtained according to the culture method of the present invention is stable. [Industrial Applicability]

[0068] The present invention provides a primary cell culture medium and a culture method for culturing human primary acute myeloid leukemia cells in vitro, and the cultured cells can be used for drug efficacy evaluation and screening. Therefore, the present invention is suitable for industrial application.

[0069] Although the present invention has been described in detail in this specification, the present invention is not limited thereto. Those skilled in the art can make modifications according to the principle of the present invention. Therefore, it should be understood that any modifications made according to the principle of the present invention fall within the protection scope of the present invention.

Claims

1. 1. A culture medium for human primary acute myeloid leukemia cells, comprising a glutamine additive, a non-essential amino acid, human interleukin-6, human interleukin-7, human interleukin-3, recombinant human FLT3 ligand, recombinant human macrophage colony-stimulating factor, and human stem cell factor; The following conditions: (1) the amount of the glutamine additive in the culture medium is 0.5 mM to 4 mM; (2) the non-essential amino acid is one or more selected from the group consisting of glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the amount of the non-essential amino acid in the culture medium is 12.5 μM to 200 μM; (3) the amount of human interleukin-6 in the culture medium is 1.89 ng / mL to 17 ng / mL; (4) the amount of human interleukin-7 in the culture medium is 1.89 ng / mL to 51 ng / mL; (5) the amount of human interleukin-3 in the culture medium is 1.89 ng / mL to 153 ng / mL; (6) the amount of the recombinant human FLT3 ligand in the culture medium is 3 ng / mL to 81 ng / mL; (7) The amount of the recombinant human macrophage colony-stimulating factor in the culture medium is 1 ng / mL to 81 ng / mL. (8) The amount of the human stem cell factor in the culture medium is 1 ng / mL to 81 ng / mL; A culture medium for human primary acute myeloid leukemia cells that satisfies all of the above.

2. The culture medium for human primary acute myeloid leukemia cells according to claim 1, further comprising an initial medium selected from the group consisting of monocyte serum-free medium and RPMI-1640, and a basal medium containing fetal bovine serum and one or more antibiotics selected from the group consisting of streptomycin / penicillin, amphotericin B, and primocin.

3. A method for culturing human primary acute myeloid leukemia cells, characterized in that the human primary acute myeloid leukemia cells are cultured using a culture medium for human primary acute myeloid leukemia cells described in claim 1 or 2.

4. 1. A method for screening or evaluating the efficacy of a drug for human primary acute myeloid leukemia, comprising: (1) culturing the human primary acute myeloid leukemia cells by the method for culturing human primary acute myeloid leukemia cells according to claim 3; (2) selecting a drug to be tested and diluting the drug into various concentration gradients; (3) adding the diluted drug to the cells obtained by culturing in step (1) and detecting cell viability; A method comprising:

Images