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

Abstract

The present invention provides a culture medium and a culture method for primary human acute myeloid leukemia cells. The primary acute myeloid leukemia cell culture medium of the present invention comprises a glutamine additive, non-essential amino acids, human interleukin-6, human interleukin-7, human interleukin-3, recombinant human FLT3Ligand, recombinant human macrophage colony-stimulating factor, and human stem cell factor. Using the culture medium and culture method of the present invention, acute myeloid leukemia cells can be cultured with higher amplification efficiency and longer in vitro culture time. The present invention also provides a method and application for human primary acute myeloid leukemia cells cultured in vitro using the culture medium for use in drug efficacy evaluation and screening.

Description

Technical Field

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

[0002] Acute myeloid leukemia (AML) is a clonal malignant proliferative blood disease of myeloid progenitor cells of the hematopoietic system. AML is an aggressive and highly heterogeneous disease with different subtypes in biology and prognosis. AML infects 1 to 5 people per 100,000 people each year and accounts for 30%-40% of all new leukemia cases. AML is the most lethal white blood cell disease with the worst prognosis and survival rate (<26% at 5 years), which has not improved since the 1970s (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).

[0003] To date, AML stem cells have been the primary focus of leukemia research because they are the leukemia subclones that acquire drug resistance and mediate relapse. This work typically requires culturing AML stem cells. Culturing AML stem cells is challenging due to the heterogeneity within the AML stem cell population and the fact that many patients' cells grow poorly, readily differentiate, and lose their ability to self-renew in vitro. Furthermore, primary AML patient samples can be difficult to obtain and, unlike transformed cell lines, do not expand indefinitely in vitro. In vivo xenograft models allow for the expansion of human AML cells but require large numbers of cells (>10 6 cells / mouse) and do not support AML cell maintenance. On the other hand, 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 assays.

[0004] Therefore, there is a need in the art for a culture medium and a culture method for rapidly expanding primary human acute myeloid leukemia cells over a long period of time without the need for co-culture with stromal cells. Summary of the Invention

[0005] In order 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.

[0006] One aspect of the present invention is to provide a culture medium for human primary acute myeloid leukemia cells, which contains a glutamine supplement, non-essential amino acids, 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).

[0007] In a preferred aspect of the present invention, the culture medium for human primary acute myeloid leukemia cells satisfies any one, multiple or all of the following conditions:

[0008] (1) The content of glutamine additive in the culture medium is preferably 0.5 mM to 4 mM;

[0009] (2) the non-essential amino acids are one or more selected from glycine, alanine, asparagine, aspartic acid, glutamic acid, proline, and serine, and the content of the non-essential amino acids in the culture medium is preferably 12.5 μM to 200 μM;

[0010] (3) The content of human IL-6 in the culture medium is preferably 1.89 ng / mL to 17 ng / mL;

[0011] (4) The content of human IL-7 in the culture medium is preferably 1.89 ng / mL to 51 ng / mL;

[0012] (5) The content of human IL-3 in the culture medium is preferably 1.89 ng / mL to 153 ng / mL;

[0013] (6) The content of human FLT3L in the culture medium is preferably 3 ng / mL to 81 ng / mL;

[0014] (7) The content of human M-CSF in the culture medium is preferably 1 ng / mL to 81 ng / mL;

[0015] (8) The content of human SCF in the culture medium is preferably 1 ng / mL to 81 ng / mL.

[0016] In another preferred embodiment, the culture medium for human primary acute myeloid leukemia cells of the present invention further comprises a basal medium containing an initial culture medium selected from a monocyte serum-free medium and RPMI-1640, 5-10% (v / v) fetal bovine serum, and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and primocin. Specifically, when streptomycin / penicillin is selected, the concentration of streptomycin is in the range of 25 μg / mL to 400 μg / mL, preferably 50 μg / mL to 200 μg / mL, and the concentration of penicillin is in the range of 25 U / mL to 400 U / mL, preferably 50 U / mL to 200 U / mL; when amphotericin B is selected, the concentration is in the range of 0.25 μg / mL to 4 μg / mL, preferably 0.5 μg / mL to 2 μg / mL; when primocin is selected, the concentration is in the range of 25 μg / mL to 400 μg / mL, preferably 50 μg / mL to 200 μg / mL.

[0017] In another aspect, the present invention also provides an in vitro culture method for primary human acute myeloid leukemia cells, comprising the step of culturing primary human acute myeloid leukemia cells in vitro using the primary human acute myeloid leukemia cell culture medium of the present invention.

[0018] In a preferred embodiment, the in vitro culture method of human primary acute myeloid leukemia cells of the present invention comprises the following steps:

[0019] 1. Isolation and Processing of Primary Human Acute Myeloid Leukemia Cells

[0020] (1) Bone marrow samples from patients with acute myeloid leukemia were centrifuged at a speed of 1200-1600 rpm for 2-6 minutes;

[0021] (2) After centrifugation, discard the upper plasma layer and add 2-3 times the volume of 1x PBS to the blood cell pellet to dilute it and mix thoroughly; add 6-8 mL of human peripheral blood lymphocyte separation solution and the diluted blood cell pellet, and centrifuge at a speed of 380-420 g, an increase speed of 1-2, a decrease speed of 0, a temperature of 20-28°C, and a centrifugation time of 25-35 minutes;

[0022] (3) After centrifugation, separate the layers in a centrifuge tube and pipette the lymphocyte layer into 3–6 mL of 1x PBS. Mix and wash the cells, then centrifuge at a speed of 1200–1600 rpm for 2–6 minutes.

[0023] (4) Discard the supernatant, add red blood cell lysis buffer to resuspend the cell pellet, and lyse for 15 to 20 minutes at a centrifugal speed of 1200 to 1600 rpm for 2 to 6 minutes;

[0024] (5) After centrifugation, discard the supernatant and add basal culture medium for later use.

[0025] 2. Cultivation using the human primary acute myeloid leukemia cell culture medium of the present invention

[0026] The primary human acute myeloid leukemia cells obtained in step 1 were resuspended in the primary human acute myeloid leukemia cell culture medium of the present invention and counted. The cell density was 1×10 5 ~4×10 6 pieces / cm 2 The cells were seeded into culture dishes and passaged until the cells in the dish were more than 90% full.

[0027] In another aspect, the cells obtained by the culture method of the present invention can be applied to regenerative medicine, basic medical research on acute myeloid leukemia cells, screening of drug responses, and development of new drugs for acute myeloid leukemia. Therefore, the present invention also provides a method for drug screening or efficacy evaluation using primary human acute myeloid leukemia cells, comprising the following steps:

[0028] (1) Cultivating primary human acute myeloid leukemia cells using the culturing method of primary human acute myeloid leukemia cells of the present invention;

[0029] (2) Select the drug to be tested and dilute it into different concentration gradients;

[0030] (3) Adding the diluted drug to the cells cultured in (1) and performing a cell activity test.

[0031] The technical solution of the present invention can achieve the following technical effects:

[0032] (1) Improve the success rate of primary human acute myeloid leukemia cell culture to over 80%;

[0033] (2) Ensure that primary human acute myeloid leukemia cells in vitro can maintain the patient's pathological characteristics;

[0034] (3) High amplification efficiency, as long as 10 5 The number of cells can be successfully expanded to 10 in about a week. 6 The expanded primary human acute myeloid leukemia cells can be continuously passaged.

[0035] (5) Controllable culture costs: the culture medium does not need to be added with expensive Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10 and other factors;

[0036] (6) The human primary acute myeloid leukemia cells cultured by the technology are large in number and highly homogenized, which are suitable for high-throughput screening of new candidate compounds and providing high-throughput in vitro drug sensitivity functional testing for patients. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 Graph showing the effects of different combinations of added factors on the proliferation of primary human acute myeloid leukemia cells.

[0038] Figures 2A-2H The graph shows the effects of different concentrations of various added factors on the proliferation of primary human acute myeloid leukemia cells.

[0039] Figure 3 The photographs are taken under a microscope of primary human acute myeloid leukemia cells cultured using the primary human acute myeloid leukemia cell culture medium of the present invention.

[0040] Figure 4 The figure shows the results of flow cytometry identification of primary human acute myeloid leukemia cells cultured using the culture medium for primary human acute myeloid leukemia cells of the present invention.

[0041] Figure 5 The figure is a cell growth curve diagram of human primary acute myeloid leukemia cells cultured in vitro using the human primary acute myeloid leukemia cell culture medium of the present invention.

[0042] Figure 6 Comparison of culture of primary human acute myeloid leukemia cells using the human primary acute myeloid leukemia cell culture medium of the present invention and the culture medium in the existing literature

[0043] Figures 7A-7F The graph is a dose-effect curve of different drugs on human primary acute myeloid leukemia cells of different generations cultured using the human primary acute myeloid leukemia cell culture medium of the present invention. DETAILED DESCRIPTION

[0044] For a better understanding of the present invention, the present invention is further described below in conjunction with embodiments and drawings. The following embodiments are merely illustrative of the present invention and are not intended to limit the present invention.

[0045] Example 1 Effects of Various Added Factors in the Culture Medium of Primary Human Acute Myeloid Leukemia Cells on the Proliferation of Primary Human Acute Myeloid Leukemia Cells

[0046] (1) Preparation of culture medium for primary human acute myeloid leukemia cells

[0047] First, prepare the basal culture medium. The basal culture medium is composed of monocyte serum-free medium (purchased from BI, 05-080-1A) + 10% (v / v) fetal bovine serum (purchased from Ecosine, FND500) + 100 μg / mL Primocin (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / mL).

[0048] Different types of growth factors (see Table 1) were added to the basal culture medium to prepare human primary acute myeloid leukemia cell culture medium containing different added components.

[0049] (2) Isolation and processing of primary human acute myeloid leukemia cells

[0050] 1. Sample selection

[0051] Bone marrow samples were obtained from AML patients by professional medical staff at specialized medical institutions, and all patients provided signed informed consent. Bone marrow samples (3–10 mL) were stored in EDTA-K2 anticoagulant tubes (manufacturer: Jiangsu Rongye) and transported refrigerated at 4–8°C.

[0052] 2. Material Preparation

[0053] After surface disinfection, place sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips in a clean bench under UV irradiation for 30 minutes. Remove 1x PBS from the 4°C refrigerator 30 minutes in advance.

[0054] 3. Sample separation

[0055] 3.1 In a laminar flow hood, pipette to mix the bone marrow sample, transfer it to a 15 mL centrifuge tube, and centrifuge at 1500 rpm for 4 minutes at room temperature.

[0056] 3.2 Add 6 mL of human peripheral blood lymphocyte separation medium (P8610, purchased from Solebol) to a new 15 mL centrifuge tube. After centrifugation of the bone marrow sample, aspirate and discard the upper plasma layer. Add 2-3 times the volume of 1x PBS to the blood cell pellet and mix thoroughly. Slowly stack the diluted blood along the wall of the centrifuge tube onto the surface of the separation medium, taking care to keep the liquid surface clear. Gently place the centrifuge tube with the bone marrow sample mixture in a centrifuge and centrifuge at 400 g for 30 minutes, with an increase speed of 2 and a decrease speed of 0 at 25°C.

[0057] 3.3 After centrifugation, separate the cells from top to bottom of the centrifuge tube into four layers (PBS layer, circular milky white lymphocyte layer, separation fluid layer, and red blood cell layer). Pipette the lymphocyte layer into a 15 mL centrifuge tube pre-filled with 5 mL of 1 x PBS. Gently mix and wash the cells. Centrifuge at 1500 rpm for 5 minutes at room temperature.

[0058] 3.4 Discard the supernatant and observe whether there are blood cells. If there are blood cells, add 8 mL of blood cell lysis buffer (purchased from Sigma, R7757-100 mL), mix well, and lyse at 4°C for 20 minutes, inverting once during the process, and centrifuge at 1500 rpm for 4 minutes at room temperature;

[0059] 3.5 Discard the supernatant and add 2 mL of basal culture medium to resuspend the cells for later use.

[0060] 4. Cell Counting and Processing

[0061] 4.1 Viable Cell Counting: 12 μL of the resuspended cell suspension was added to 12 μL of trypan blue dye (Shanghai Sangon Biotech Co., Ltd.). After thorough mixing, 20 μL was added to a cell counting plate (Countstar, specification: 50 plates / box). The percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) as follows: number of viable cells / total number of cells*100%.

[0062] (3) Culture of primary human acute myeloid leukemia cells

[0063] The culture medium with different components in Table 1 was added to a 96-well plate at a volume of 100 μL / well. Human primary acute myeloid leukemia cells were isolated from two human primary acute myeloid leukemia bone marrow samples (numbered A17007 and A25104, from the First Affiliated Hospital of Anhui Medical University) according to the above step (2) and cultured at a volume of 1×10 4 Cells were seeded at a density of 1000 cells / well in a 96-well culture plate and cultured at 37°C and 5% CO2. After 5-8 days of culture, when the cells had grown to 70-85%, 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 2-4 hours. Mix well and read the plate at 450 nm using a multi-mode microplate reader (Multi-Mode Detection Platform, Molecular Instruments (Shanghai) Co., Ltd., USA). As an experimental control, a basal culture medium without any additives was used. The experimental results are shown in Table 1.

[0064] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0065]

[0066]

[0067] Among them, "+" indicates that compared with the basal medium, the culture medium to which the additive is added has a proliferation-promoting effect on two human primary acute myeloid leukemia cells isolated from primary human acute myeloid leukemia bone marrow samples; "-" indicates that the culture medium to which the additive is added has a proliferation-promoting effect on at least one human primary acute myeloid leukemia cell isolated from primary human acute myeloid leukemia bone marrow samples; and "○" indicates that the culture medium to which the additive is added has no significant effect on the proliferation of at least two human primary acute myeloid leukemia cells isolated from primary human acute myeloid leukemia bone marrow samples.

[0068] Based on the above results, human IL-7, human IFN-α, human M-CSF, human SCF, human IL-6, glutamine additive, human FLT3L, human IL-3, and non-essential amino acids were selected for further culture experiments in Example 2.

[0069] Example 2 Effects of different combinations of added factors in the culture medium of primary human acute myeloid leukemia cells on the proliferation of primary human acute myeloid leukemia cells

[0070] According to the components in Table 2, culture media for human primary acute myeloid leukemia cells with different combinations of added factors were prepared to investigate the proliferation-promoting effects of different combinations of added factors on human primary acute myeloid leukemia cells.

[0071] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0072]

[0073] According to the method of step (2) 3 of Example 1, 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). The obtained cell suspension was divided into 11 equal parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and No. 1 to 10 culture media were used to resuspend the cells at a viable cell density of 2×10 4 Cells were seeded into 48-well plates (20,000 cells per well), and the volume of each well in the 48-well plate was filled to 1 mL with the corresponding culture medium. The cells were mixed thoroughly. After surface disinfection, the cells were cultured in a 37°C, 5% CO2 incubator (purchased from Thermo Fisher Scientific).

[0074] When cells in a 48-well plate grew to over 85%, they were transferred to a 15 mL centrifuge tube and centrifuged at 1500 rpm for 5 minutes. The cell pellet was resuspended in 500 μL of monocyte serum-free medium. 12 μL of the resuspended cell suspension was added to 12 μL of trypan blue stain (Shanghai Sangon Biotech Co., Ltd.) and thoroughly mixed. 20 μL was then added to a cell counting plate (Countstar, specification: 50 plates / box). The percentage of viable large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) as follows: number of viable cells / total number of cells x 100%. The results obtained for primary human acute myeloid leukemia cells from bone marrow samples A17151, A17152, A20090, A20101, A21004, and A20141 are shown in Figure 2. Figure 1 .

[0075] according to Figure 1 The results show that compared with the basal culture medium, the use of the above-mentioned No. 1 to No. 10 culture media can promote the proliferation of primary intestinal cancer cells to varying degrees. The culture medium formula No. 2 does not contain human IFN-α, but instead leads to a better proliferation effect. The results show that factors such as glutamine additives, human SCF, human IL-6, human IL-3, human FLT3L, non-essential amino acids, human IL-7 and human M-CSF have a significant pro-proliferation effect on human primary acute myeloid leukemia cells.

[0076] Example 3 Proliferation-promoting effects of different concentrations of added factors on primary human acute myeloid leukemia cells

[0077] Primary human acute myeloid leukemia cells were obtained from primary human acute myeloid leukemia bone marrow samples (numbers A23065 and A17112) according to the method of step (2)-3 of Example 1. The cells were resuspended in basal culture medium (monocyte serum-free medium + 10% (v / v) fetal bovine serum + 100 μg / mL Primocin) and set aside.

[0078] Next, the factor having a cell culture proliferation effect determined in Example 2 (prepared according to Formula No. 2 in Example 2) was added to the basal medium to form a combined basal medium. The following 8 culture medium formulas were then prepared for the experiment:

[0079] Formulation 1: The combined basic medium components do not contain glutamine additive;

[0080] Formulation 2: Combination of basic medium components without human SCF;

[0081] Formulation 3: Combination of basal medium components without human IL-6;

[0082] Formulation 4: Combination of basic medium components without human IL-3;

[0083] Formulation 5: Combination of basic medium components without human FLT3L;

[0084] Formulation 6: The combined basic medium components do not contain non-essential amino acids;

[0085] Formulation 7: Combination of basic medium components without human IL-7;

[0086] Formulation 8: Combination of basic culture medium components without human M-CSF;

[0087] Add 20 μl of 4x10 4 For each cell resuspension, 1 mL of the culture medium of formulas 1 to 8 was used to dilute the cell suspension.

[0088] When using the culture medium of Formula 1, add 1 mL of the prepared glutamine additive to each well of a 48-well plate seeded with primary cells. The final concentrations of the glutamine additive are 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM, respectively. Set up control wells (BC) using the culture medium of Formula 1.

[0089] When using the medium of Formula 2, add 1 mL of prepared human SCF to each well of a 48-well plate seeded with primary cells. The final concentrations of human SCF are 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Set up control wells (BC) using the medium of Formula 2.

[0090] When using the culture medium of Formula 3, 1 mL of prepared human IL-6 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human IL-6 were 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 3.

[0091] When using the culture medium of Formula 4, 1 mL of prepared human IL-3 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human IL-3 were 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 4.

[0092] When using the culture medium of Formula 5, 1 mL of prepared human FLT3L was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human FLT3L were 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 5.

[0093] When using the culture medium of Formula 6, 1 mL of the prepared non-essential amino acids was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the non-essential amino acids were 12.5 μM, 25 μM, 50 μM, 100 μM, and 200 μM, respectively. Control wells (BC) were set up using the culture medium of Formula 6.

[0094] When using the culture medium of Formula 7, 1 mL of prepared human IL-7 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human IL-7 were 1.89 ng / mL, 5.67 ng / mL, 17 ng / mL, 51 ng / mL, and 153 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 7.

[0095] When using the medium of Formula 8, add 1 mL of prepared human M-CSF to each well of a 48-well plate seeded with primary cells. The final concentrations of human M-CSF are 1 ng / mL, 3 ng / mL, 9 ng / mL, 27 ng / mL, and 81 ng / mL, respectively. Set up control wells (BC) using the medium of Formula 8.

[0096] When the cells have expanded to about 85% of the 48 wells, count them and calculate the proliferation times by referring to the number of cells in the control wells (BC). The results are shown in Figures 2A to 2H . Figures 2A to 2H The ratio is the ratio of the number of cells cultured in each culture medium for one generation to the number of cells cultured in the corresponding control well for one generation. A ratio greater than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation more effectively than the culture medium in the control well. A ratio less than 1 indicates that the culture medium containing the factor or small molecule compound at different concentrations promotes cell proliferation less effectively than the culture medium in the control well.

[0097] according to Figures 2A to 2HThe results show that 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 proliferative effect on human primary acute myeloid leukemia cells. According to the results of this embodiment, the content of glutamine additive is preferably 0.5mM to 4mM, and the concentration of 0.5mM is the most obvious when added to cell proliferation; the content of human SCF is preferably 1ng / ml to 81ng / ml, and the concentration of 9ng / mL is the most obvious when added to cell proliferation; the content of human IL-6 is preferably 1.89ng / ml to 17ng / ml, and the concentration of 5.67ng / mL is the most obvious when added to cell proliferation; the content of human IL-3 is preferably 1.89ng / ml to 153ng / ml, and the concentration of 51ng / mL is the most obvious when added to cell proliferation. The effect is most obvious; the content of human FLT3L is preferably 3ng / ml~81ng / ml, and the concentration is 27ng / mL, and the most obvious cell proliferation effect is achieved when it is added to the cell; the content of non-essential amino acids is preferably 12.5μM~200μM, and the most obvious cell proliferation effect is achieved when it is added to the cell at a concentration of 50μM; the content of human IL-7 is preferably 1.89ng / ml~51ng / ml, and the most obvious cell proliferation effect is achieved when it is added to the cell at a concentration of 17ng / mL; the content of human M-CSF is preferably 1ng / ml~81ng / ml, and the most obvious cell proliferation effect is achieved when it is added to the cell at a concentration of 27ng / mL.

[0098] Example 4 Culture and identification of primary human acute myeloid leukemia cells

[0099] (1) Culture of primary human acute myeloid leukemia cells

[0100] Human primary acute myeloid leukemia cells were obtained from a human primary acute myeloid leukemia bone marrow sample (numbered A17030) according to the method of step (2) of Example 1, and cultured using the human primary acute myeloid leukemia cell culture medium of the present invention (composed of the optimal composition and concentration combination determined in Example 3, i.e., comprising basal medium, 0.5 mM glutamine additive, 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 viable cell density of 3×10 6 Cells were seeded into 6-well plates, 5 mL of the human primary acute myeloid leukemia cell culture medium of the present invention was added and mixed evenly, and the cells were placed in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific) for culture after surface disinfection.

[0101] The cultured human primary acute myeloid leukemia cells were observed using a microscope (Invitrogen EVOS M500). Figure 3 The images were taken at 10x magnification on day 1, day 4, and day 7 of culture. Cell counts showed a 3.58-fold increase in viable cells after day 7 of culture.

[0102] (2) Flow cytometry identification of primary human acute myeloid leukemia cells

[0103] According to the method of step (2) of Example 1, human primary acute myeloid leukemia cells were obtained from a human primary acute myeloid leukemia bone marrow sample (numbered A17014), and cultured using the human primary acute myeloid leukemia cell culture medium of the present invention. Specifically, the obtained human primary acute myeloid leukemia cells were cultured at a viable cell density of 1×10 6 Each well of the plate was inoculated with 12 cells, 3 mL of the culture medium of the present invention was added and mixed, and the plate was placed in a 37° C., 5% CO 2 incubator (purchased from Thermo Fisher Scientific) for culture after surface disinfection.

[0104] Human primary acute myeloid leukemia cells before culture and after 7 days of culture were transferred to 15 mL centrifuge tubes and centrifuged at 1500 rpm at room temperature for 5 minutes. The supernatant was discarded, and the cell pellet was diluted with 2 mL of 1x PBS and divided equally into two 1.5 mL centrifuge tubes. One portion was used for double labeling of leukocytes (APC Mouse Anti-Human CD45 (purchased from BD, 560973)) and myeloid markers (BB515 Mouse Anti-Human CD33 (purchased from BD, 564588)) in the experimental group; the other portion was used as a blank control group. The cells were centrifuged at 1500 rpm at room temperature for 5 minutes, the supernatant was discarded, 40 μL of 0.5% BSA (prepared in 1X PBS) was added, and the above-mentioned antibodies were added at a 1:40 ratio in the dark. No antibodies were added to the control group, the cells were mixed, and the cells were incubated on ice for 1-2 hours. After the incubation, 1 mL of 1X PBS was added to each tube for resuspending and washing, and the cells were centrifuged at 1500 rpm at room temperature for 5 minutes. The supernatant was discarded, and the cell pellet was resuspended in 300 μL of 1X PBS. The expression of myeloid markers in primary human acute myeloid leukemia cells before and after 7 days of culture was analyzed using a flow cytometer (Beckman EVOS M500).

[0105] Figure 4 This is the result of flow cytometry identification of human primary acute myeloid leukemia cells cultured using the human primary acute myeloid leukemia cell culture medium of the present invention. Figure 4 It was confirmed that the proportion of myeloid leukemia cells increased by 16% after the primary human acute myeloid leukemia cells were cultured in the medium of the present invention for 7 days.

[0106] Example 5 Primary Culture Cycle and Cell Number Statistics of Human Primary Acute Myeloid Leukemia Cells and Calculation of Population Doubling (PD) Values

[0107] According to the method of step (2) 3 of Example 1, human primary acute myeloid leukemia cells were obtained from 8 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 a viable cell density of 1×10 6 Cells were seeded in 12-well plates at 400 μg / well and cultured using the culture medium of the present invention. After 5 to 9 days of cell culture, the cells were passaged and counted, and the number of days in culture until passage was recorded. The number of days in culture until passage was taken as a culture cycle. Under the experimental conditions, the cells obtained by amplification were amplified for different generations. After each generation, the cells were counted and the corresponding culture cycles were recorded. PD was calculated according to the formula Population Doubling (PD) = 3.32 * log10 (total number of cells after digestion / initial number of cells seeded). For the formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

[0108] Figure 5 The growth curves of 8 primary cells cultured using the human primary acute myeloid leukemia cell base of the present invention, drawn using Graphpad Prism software, are shown. The horizontal axis represents the number of days of cell culture, and the vertical axis is the cumulative cell proliferation multiple, which represents the multiple of cell expansion during the culture cycle. The larger the value, the more times the cells expand within a certain period, that is, the more cells are expanded. The slope represents the rate of cell expansion. Figure 5 It can be confirmed that when the human primary acute myeloid leukemia cells cultured in the culture medium of the present invention are continuously cultured and expanded for at least 45 days, the cell expansion rate remains substantially unchanged and still has the ability to continue to expand.

[0109] Comparison of Example 6 with existing culture medium culture effects

[0110] (1) Preparation of control culture medium

[0111] The control culture medium was prepared according to the literature (Silvia Ravera et al., Scientific Reports, (2020) 10: 16519), whose formula is 1640 culture medium (purchased from Corning, 10-040-CVR) + 10 ng / mL IL-15 (purchased from SinoBiological) + 10 ng / mL IL-4 (purchased from Sino Biological) + 10% FBS (purchased from excellbio, FND500) (hereinafter referred to as "control culture medium").

[0112] (2) Acquisition and culture of primary human acute myeloid leukemia cells

[0113] Human primary acute myeloid leukemia cells were obtained from a primary human acute myeloid leukemia bone marrow sample (A10093) according to the method of step (2) of Example 1. The viable cell density was 1×10 6 Each well was inoculated into a 12-well plate and cultured with the culture medium of the present invention and the control culture medium respectively.

[0114] On the 7th day of culture, the 12-well plate was removed and the cell suspension was transferred to a 15 mL centrifuge tube. The cells were centrifuged at 1500 rpm for 5 minutes at room temperature and the cell pellet was resuspended in 1 mL of culture medium. 12 μL of the resuspended cell suspension was taken and mixed thoroughly with 12 μL of trypan blue dye (Sangon Biotech (Shanghai) Co., Ltd.). 20 μL was then added to a cell counting plate (Countstar, specification: 50 plates / box). The total number of cells was counted using a cell counter (Countstar, IC1000). The count results were displayed on Figure 6 .

[0115] according to Figure 6 The results show that, compared with the control culture medium, the human primary acute myeloid leukemia culture medium of the present invention can significantly promote the expansion of human primary acute myeloid leukemia cells, and its effect is better than that of the control culture medium.

[0116] Example 7: Primary human acute myeloid leukemia cells expanded using the culture medium of the present invention are used for drug screening and efficacy evaluation

[0117] 1. Cell Culture and Plating

[0118] Human primary acute myeloid leukemia cells (numbered A23170) were isolated and cultured using the human primary acute myeloid leukemia cell culture medium of the present invention. After the cells expanded to 85%, they were passaged. The cell passages were counted according to the steps in Example 1 and the cells were plated at a viable cell density of 1×10 5 After the cells / mL were placed in a sample addition tank (purchased from Corning) and fully mixed, they were cultured in a 384-well opaque white cell culture plate (purchased from Corning), with a volume of 50 μL per well and 5000 cells / well. The human primary acute myeloid leukemia culture medium of the present invention was added from the edge of the well plate to seal the plate, and the sample name, dosing time and CellTiter-Glo (purchased from Promega) detection time were marked on the plate. The surface was disinfected with 75% alcohol (purchased from Lierkang), cultured in a 37°C, 5% CO2 incubator, and the drug was added after 24 hours. The first, second, third, fourth and fifth generation cells were obtained for drug screening, and the drug sensitivity of the primary cells cultured using the culture medium of the present invention was tested.

[0119] 2. Screening drug preparation

[0120] Six drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, homoharringtonine; all purchased from MCE) with six concentration gradients were prepared according to the table below, 30 μL was added to each well of a 384-well drug plate (purchased from Thermo Fisher Scientific), and stored for use.

[0121] Table 3 Drug action concentration settings

[0122]

[0123] 3. High-throughput dosing

[0124] Remove the prepared drug plate, place it at room temperature, and centrifuge it in a Beckman centrifuge at 1000 rpm for 1 minute. High-throughput drug addition was performed using a high-throughput automated sample delivery system (Perkin Elmer JANUS). 0.1 μL of the selected drug at the corresponding concentration was added to each well of a 384-well plate containing primary human acute myeloid leukemia cells. After drug addition, the 384-well plate was surface-disinfected and moved to an incubator. Cell viability was measured 72 hours later.

[0125] 4. Cell activity test

[0126] Remove CellTiter-Glo luminescent reagent (purchased from Promega) from a 4°C refrigerator and place 10 mL of the reagent in a sample reservoir. Remove the 384-well plate to be tested from the incubator and add 10 μL of CellTiter-Glo luminescent reagent to each well. Let it stand for 10 minutes, then mix thoroughly and analyze using a multi-function microplate reader (Perkin Elmer Envision).

[0127] 5. Data processing

[0128] The cell inhibition rate after different drugs acted on cells was calculated according to the formula: cell inhibition rate (%) = 100% - chemiluminescence value of drug-treated wells / chemiluminescence value of control wells * 100%. The half inhibition rate (IC) of drug action on cells was calculated using GraphPad Prism software. 50 The results are shown in Figures 7A-7F .

[0129] Depend on Figures 7A-7FIt can be confirmed that the human primary acute myeloid leukemia cells obtained by culturing the human primary acute myeloid leukemia cell culture medium of the present invention were subjected to drug screening, and the inhibitory effect of the same drug on cells of different generations of culture remained basically consistent (the inhibition curve remained basically consistent). The cells of the same patient had different sensitivities to different drugs at the maximum blood drug concentration in the human body. Based on the results, the effectiveness of the drug in clinical use of the primary acute myeloid leukemia patients can be judged, and it can be shown that the sensitivity of tumor cells of different generations obtained according to the culture method of this patent is stable to the drug.

[0130] Industrial Applicability

[0131] The present invention provides a primary cell culture medium and a culture method for culturing primary human acute myeloid leukemia cells in vitro. The cultured cells can be used for evaluating and screening drug efficacy. Therefore, the present invention is suitable for industrial application.

[0132] Although the present invention is described in detail herein, the present invention is not limited thereto. Those skilled in the art may make modifications based on the principles of the present invention. Therefore, all modifications made in accordance with the principles of the present invention should be understood to fall within the scope of protection of the present invention.

Claims

1. A culture medium for primary human acute myeloid leukemia cells, characterized in that: Made with the following ingredients: Glutamine supplement; non-essential amino acids; human interleukin-6; human interleukin-7; human interleukin-3; recombinant human FLT3Ligand; recombinant human macrophage colony stimulating factor; human stem cell factor; an initial culture medium selected from a monocyte serum-free medium or RPMI-1640; fetal bovine serum; and one or more antibiotics selected from streptomycin / penicillin, amphotericin B, and primocin, wherein: (1) The content 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 glycine, alanine, asparagine, aspartic acid, glutamic acid, proline and serine, and the content of the non-essential amino acid in the culture medium is 12.5 μM to 200 μM; (3) The content of human interleukin-6 in the culture medium is 1.89 ng / mL to 17 ng / mL; (4) The content of human interleukin-7 in the culture medium is 1.89 ng / mL to 51 ng / mL; (5) The content of human interleukin-3 in the culture medium is 1.89 ng / mL to 153 ng / mL; (6) The content of the recombinant human FLT3 Ligand in the culture medium is 3 ng / mL to 81 ng / mL; (7) The content of the recombinant human macrophage colony stimulating factor in the culture medium is 1 ng / mL to 81 ng / mL; (8) The content of the human stem cell factor in the culture medium is 1 ng / mL to 81 ng / mL.

2. A method for culturing primary human acute myeloid leukemia cells, characterized in that: The primary human acute myeloid leukemia cells are cultured using the primary human acute myeloid leukemia cell culture medium as claimed in claim 1.

3. A drug screening method for human primary acute myeloid leukemia, comprising the following steps: (1) Cultivating primary human acute myeloid leukemia cells using the method for culturing primary human acute myeloid leukemia cells as described in claim 2; (2) Select the drug to be tested and dilute it into different concentration gradients; (3) Add the diluted drug to the cells cultured in (1) and perform a cell activity test.

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