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

Abstract

The present invention provides a culture medium and a culture method for primary human acute lymphoblastic leukemia cells. The acute lymphoblastic leukemia cell culture medium of the present invention comprises a glutamine additive, non-essential amino acids, recombinant human FLT3 ligand, human interleukin-3, human interleukin-7, human interleukin-2, human stem cell factor, and human thrombopoietin. Using the culture medium and culture method of the present invention, acute lymphoblastic leukemia cells can be cultured with higher amplification efficiency and longer in vitro culture time. The present invention also provides methods and applications for using the culture medium to culture primary human acute lymphoblastic leukemia cells in vitro for 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 lymphoblastic leukemia cells in vitro, a method for culturing primary human acute lymphoblastic leukemia cells in vitro using the culture medium, and an application of the culture medium in evaluating and screening drug efficacy. Background Art

[0002] Acute lymphoblastic leukemia (ALL) is a malignant tumor disease that originates from the abnormal proliferation of B or T lymphocytes in the bone marrow. The abnormally proliferating primitive cells can accumulate in the bone marrow and inhibit normal hematopoietic function. They can also invade tissues outside the bone marrow, such as the meninges, lymph nodes, gonads, and liver. my country has conducted a survey on the incidence of leukemia, and the incidence of ALL is approximately 0.67 per 100,000. The peak incidence of ALL is in childhood (0 to 9 years old), accounting for more than 70% of childhood leukemia. In adults, ALL accounts for approximately 20% of adult leukemia. Currently, the prognosis for minors with ALL is good, but in adults, the prognosis is poor and the survival rate is low. Therefore, new and effective treatments need to be developed for ALL.

[0003] To date, ALL-related research has primarily relied on ALL cell lines, which are prone to differentiation and apoptosis during long-term culture. Patient-derived ALL sample cells can be expanded and proliferated by xenotransplantation into immunodeficient mice for in vivo experiments. However, in vivo studies are expensive and time-consuming, and the human and material resources required are unpredictable, especially for novel treatments or drug screening experiments. Therefore, ALL-related research requires the in vitro culture of primary ALL cells.

[0004] On the other hand, in vitro culture of primary human acute lymphoblastic leukemia cells relies on co-culture with stromal cells, but cultures of bone marrow (BM)-derived mesenchymal stromal cells (MSCs) or stromal cell lines are not suitable for high-throughput drug screening or adaptive assays.

[0005] Therefore, there is a need in the art for a culture medium for rapidly expanding primary human acute lymphoblastic leukemia cells over a long period of time without the need for co-culture with stromal cells. Summary of the Invention

[0006] 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 lymphoblastic leukemia cells in vitro.

[0007] One aspect of the present invention is to provide a culture medium for human primary acute lymphoblastic leukemia cells, which contains glutamine supplement, non-essential amino acids, recombinant human FLT3Ligand (human FLT3L), human interleukin-3 (human IL-3), human interleukin-7 (human IL-7), human interleukin-2 (human IL-2), human stem cell factor (human SCF), and human thrombopoietin (human TPO).

[0008] In a preferred aspect of the present invention, the culture medium for human primary acute lymphoblastic leukemia cells satisfies any one, multiple or all of the following:

[0009] (1) 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 preferably 12.5 μM to 200 μM, more preferably 12.5 μM to 50 μM;

[0010] (2) The content of the glutamine additive in the culture medium is preferably 1 mM to 2 mM;

[0011] (3) The content of human FLT3L in the culture medium is preferably 20 ng / ml to 320 ng / ml, more preferably 80 ng / mL to 320 ng / mL;

[0012] (4) The content of human IL-3 in the culture medium is preferably 1.89 ng / ml to 153 ng / ml, more preferably 5.67 ng / mL to 153 ng / mL;

[0013] (5) The content of human IL-7 in the culture medium is preferably 1 ng / ml to 81 ng / ml, more preferably 3 ng / ml to 81 ng / ml;

[0014] (6) The content of human IL-2 in the culture medium is preferably 1 ng / ml to 81 ng / ml, more preferably 9 ng / mL to 81 ng / mL;

[0015] (7) The content of human SCF in the culture medium is preferably 1 ng / ml to 81 ng / ml, more preferably 9 ng / mL to 81 ng / mL;

[0016] (8) The content of human TPO in the culture medium is preferably 1 ng / ml to 81 ng / ml, more preferably 1 ng / ml to 9 ng / mL.

[0017] In another preferred embodiment, the culture medium for human primary acute lymphoblastic 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.

[0018] In another aspect, the present invention further provides an in vitro culture method for primary human acute lymphoblastic leukemia cells, comprising culturing primary human acute lymphoblastic leukemia cells in vitro using the primary human acute lymphoblastic leukemia cell culture medium of the present invention.

[0019] The in vitro culture method of human primary acute lymphoblastic leukemia cells of the present invention further comprises the following steps.

[0020] 1. Isolation and Processing of Primary Human Acute Lymphoblastic Leukemia Cells

[0021] (1) Bone marrow samples from patients with acute lymphoblastic leukemia were centrifuged at a speed of 1200 to 1600 rpm for 2 to 6 minutes;

[0022] (2) Prepare a discontinuous density gradient Percoll layer by using physiological saline to prepare Percoll separation solutions of different densities, and place the Percoll separation solutions of different densities layer by layer from high density to low density;

[0023] (3) After centrifugation, the upper plasma layer was discarded, and 2-3 times the volume of physiological saline was added to the blood cell pellet to dilute it and mix thoroughly. The diluted blood cell pellet was added to the discontinuous density gradient Percoll layer and centrifuged at 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.

[0024] (4) After centrifugation, circularly pipette the lymphocyte layer into physiological saline, gently mix and wash the cells, and then centrifuge at a speed of 1200-1600 rpm for 2-6 minutes;

[0025] (5) Discard the supernatant, add red blood cell lysis buffer to resuspend the cell pellet, lyse for 15-20 minutes, and centrifuge at 1200-1600 rpm at room temperature for 2-6 minutes;

[0026] (6) After centrifugation, discard the supernatant and add basal culture medium for later use.

[0027] 2. Cultivation using the human primary acute lymphoblastic leukemia cell culture medium of the present invention

[0028] The primary human acute lymphoblastic leukemia cells obtained in step 1 were resuspended in the primary human acute lymphoblastic leukemia cell culture medium of the present invention and counted according to the cell density of 1 to 4×10 5 pieces / cm 2 The cells were seeded into culture dishes and passaged until the cells in the dish were more than 90% full.

[0029] 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 lymphoblastic leukemia cells, screening of drug responses, and development of new drugs for acute lymphoblastic leukemia. Therefore, the present invention also provides a method for drug screening or efficacy evaluation for human primary acute lymphoblastic leukemia, comprising the following steps:

[0030] (1) Cultivating primary human acute lymphoblastic leukemia cells using the culturing method of primary human acute lymphoblastic leukemia cells of the present invention;

[0031] (2) Select the drug to be tested and dilute it into different concentration gradients;

[0032] (3) Adding the diluted drug to the cells cultured in (1) and performing a cell activity test.

[0033] The technical solution of the present invention can achieve the following technical effects:

[0034] (1) Improve the success rate of primary human acute lymphoblastic leukemia cell culture to over 80%;

[0035] (2) Ensure that primary human acute lymphoblastic leukemia cells in vitro can maintain the patient's pathological characteristics;

[0036] (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 lymphoblastic leukemia cells can be continuously passaged.

[0037] (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;

[0038] (6) The human primary acute lymphoblastic leukemia cells cultured using the technology are large in number and highly homogenized, making them 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

[0039] Figure 1 Graph showing the effects of different combinations of added factors on the proliferation of primary human acute lymphoblastic leukemia cells.

[0040] Figures 2A-2H The graph shows the effect of different concentrations of various added factors on the proliferation of primary human acute lymphoblastic leukemia cells.

[0041] Figure 3 The photographs are taken under a microscope of primary human acute lymphoblastic leukemia cells cultured using the primary human acute lymphoblastic leukemia cell culture medium of the present invention.

[0042] Figures 4A-4C The figure shows the results of flow cytometry identification of primary human acute lymphoblastic leukemia cells cultured using the culture medium for primary human acute lymphoblastic leukemia cells of the present invention.

[0043] Figure 5 The figure is a cell growth curve diagram of human primary acute lymphoblastic leukemia cells cultured in vitro using the human primary acute lymphoblastic leukemia cell culture medium of the present invention.

[0044] Figure 6 The figure is a comparison of culturing primary human acute lymphoblastic leukemia cells using the culture medium of the present invention and the culture medium in the existing literature.

[0045] Figures 7A-7F The graph is a dose-effect curve of different drugs on human primary acute lymphoblastic leukemia cells of different generations cultured using the human primary acute lymphoblastic leukemia cell culture medium of the present invention. DETAILED DESCRIPTION

[0046] 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.

[0047] Example 1 Effects of various added factors in the culture medium of primary human acute lymphoblastic leukemia cells on the proliferation of primary human acute lymphoblastic leukemia cells

[0048] (1) Preparation of culture medium for primary human acute lymphoblastic leukemia cells

[0049] 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).

[0050] Different types of growth factors (see Table 1) were added to the basal culture medium to prepare human primary acute lymphoblastic leukemia cell culture medium containing different added components.

[0051] (2) Isolation and processing of primary human acute lymphoblastic leukemia cells

[0052] 1. Sample selection

[0053] Bone marrow samples were obtained from ALL patients by professional medical staff at specialized medical institutions, with informed consent from all patients. Bone marrow samples (3–10 mL) were stored in EDTA-K2 anticoagulant tubes (manufacturer: Jiangsu Rongye) and transported refrigerated at 4–8°C.

[0054] 2. Material Preparation

[0055] After surface disinfection, sterile 15mL centrifuge tubes, pipettes, 10mL pipettes, and sterile pipette tips should be placed in a clean bench under UV irradiation for 30 minutes. Remove 8.5% saline from a 4°C refrigerator 30 minutes in advance.

[0056] 3. Sample separation

[0057] 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.

[0058] 3.2 Prepare a discontinuous density gradient Percoll layer. Prepare 2 mL of each of 70% (specific gravity 1.090 g / L), 60% (specific gravity 1.077 g / L), 50% (specific gravity 1.067 g / L), and 40% (specific gravity 1.056 g / L) Percoll separation solutions in 8.5% saline in a 15 mL centrifuge tube. In another 15 mL centrifuge tube, pipette the Percoll separation solutions of different densities from high density to low density and slowly place them layer by layer along the tube wall.

[0059] 3.3 After centrifugation, discard the upper plasma layer and dilute the blood cell pellet with 2-3 times the volume of 8.5% saline and mix thoroughly. Slowly add the diluted blood cell pellet to the discontinuous density gradient Percoll layer, until it is clearly separated from the separation solution. Centrifuge at 400g, 2% ramp, 0% ramp, and 25°C for 30 minutes.

[0060] 3.4 After centrifugation, pipette the lymphocyte layer into a 15 mL centrifuge tube pre-filled with 5 mL of 8.5% saline. Gently mix and wash the cells, then centrifuge at 1500 rpm for 5 minutes.

[0061] 3.5 Discard the supernatant and resuspend the cell pellet in 6 mL of red blood cell lysis buffer (purchased from Sigma). Lyse the pellet at 4°C for 15-20 minutes, inverting once to mix thoroughly. Centrifuge at 1500 rpm for 5 minutes at room temperature.

[0062] 3.6 After centrifugation, discard the supernatant and add 2 mL of basal culture medium for later use.

[0063] 4. Cell Counting and Processing

[0064] 12 μL of the resuspended cell suspension was added to 12 μL of trypan blue dye (Shanghai Sangon Biotech Co., Ltd.), mixed thoroughly, and 20 μL was added to a cell counting plate (Countstar, specification: 50 plates / box). The percentage of live large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) = number of live cells / total number of cells*100%.

[0065] (3) Culture of primary human acute lymphoblastic leukemia cells

[0066] 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 lymphoblastic leukemia cells were isolated from two human primary acute lymphoblastic leukemia bone marrow samples (numbered A17008 and A10100, from the First Affiliated Hospital of Anhui Medical University) according to the above step (2) and cultured at 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.

[0067] Table 1 Additives in culture medium and their effects on promoting cell proliferation

[0068]

[0069] Among them, "+" indicates that compared with the basal medium, the culture medium adding the additive has a proliferation-promoting effect on two human primary acute lymphoblastic leukemia cells isolated from primary human acute lymphoblastic leukemia bone marrow samples; "-" indicates that the culture medium adding the additive has a proliferation-promoting effect on one human primary acute lymphoblastic leukemia cell isolated from primary human acute lymphoblastic leukemia bone marrow samples; "○" indicates that the culture medium adding the additive has no obvious effect on the proliferation of two human primary acute lymphoblastic leukemia cells isolated from primary human acute lymphoblastic leukemia bone marrow samples.

[0070] Based on the above results, factors such as human M-CSF, glutamine additive, human SCF, human FLT3L, human IL-3, and non-essential amino acids were selected to carry out the culture experiment of Example 2.

[0071] Example 2 Effects of different combinations of added factors in the culture medium of primary human acute lymphoblastic leukemia cells on the proliferation of primary human acute lymphoblastic leukemia cells

[0072] According to the components in Table 2, culture media for human primary acute lymphoblastic 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 lymphoblastic leukemia cells.

[0073] Table 2 Preparation of different components of culture medium (concentration is final concentration)

[0074]

[0075] Human primary acute lymphoblastic leukemia cells were obtained from primary human acute lymphoblastic leukemia bone marrow samples (numbered A20092, A20145, and A20142) according to the method of step (2) of Example 1. The obtained cell suspension was divided into 8 equal parts and centrifuged at 1500 rpm for 4 minutes. After centrifugation, 200 μL of BM and No. 1 to 7 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).

[0076] 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 live large cells (cell size >10 μm) was calculated using a cell counter (Countstar, IC1000) as follows: number of live cells / total number of cells x 100%. The results obtained for primary human acute lymphoblastic leukemia cells from bone marrow samples A20092, A20145, and A20142 are shown in Figure 2. Figure 1 .

[0077] according to Figure 1 The results showed that, compared with the basal medium, the above No.1 to No.7 mediums were able to promote the proliferation of primary intestinal cancer cells to varying degrees. Figure 1 The results showed that glutamine supplement, human FLT3L, human IL-3, human SCF, and non-essential amino acids had a significant proliferative effect on primary human acute lymphoblastic leukemia cells, while human M-CSF had no significant effect on the culture of primary human acute lymphoblastic leukemia cells.

[0078] Example 3 Proliferation-promoting effects of different concentrations of added factors on primary human acute lymphoblastic leukemia cells

[0079] Primary human acute lymphoblastic leukemia cells were obtained from primary human acute lymphoblastic leukemia bone marrow samples (numbers A17010, A17113, A23194, A13047, A23082, and A23177) 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 (purchased from InvivoGen, 0.2% (v / v), commercially available at a concentration of 50 mg / mL)) and set aside.

[0080] Next, the factors with cell culture proliferation effects identified in Example 2 (glutamine supplement, human FLT3L, human IL-3, human SCF, and non-essential amino acids) and the factors with a significant proliferation effect on one case identified in Example 1 (human IL-7, human IL-2, human TPO) were added to the basal medium to form a combined basal medium. The following eight culture medium formulations were then prepared for the experiment:

[0081] Formulation 1: Combination of basic medium components without human IL-7;

[0082] Formulation 2: Combination of basic medium components without human IL-2;

[0083] Formulation 3: Combination of basic medium components without human SCF;

[0084] Formulation 4: The combined basic medium components do not contain non-essential amino acids;

[0085] Formulation 5: Combination of basic medium components without human TPO;

[0086] Formulation 6: The combined basic medium components do not contain glutamine additive;

[0087] Formulation 7: Combination of basic medium components without human IL-3;

[0088] Formulation 8: Combined basal medium components without human FLT3L.

[0089] Add 20 μL of 4 x 10 4 For each cell resuspension, 1 mL of the culture medium of formulas 1 to 8 was used to dilute the cell suspension.

[0090] When using the culture medium of Formula 1, 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 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 1.

[0091] When using the culture medium of Formula 2, 1 mL of prepared human IL-2 was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human IL-2 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 2.

[0092] When using the medium of Formula 3, 1 mL of prepared human SCF was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human SCF 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 medium of Formula 3.

[0093] When using the culture medium of Formula 4, 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 4.

[0094] When using the culture medium of Formula 5, 1 mL of prepared human TPO was added to each well of a 48-well plate seeded with primary cells. The final concentrations of human TPO 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.

[0095] When using the culture medium of Formula 6, 1 mL of the prepared glutamine additive was added to each well of a 48-well plate seeded with primary cells. The final concentrations of the glutamine additive were 0.5 mM, 1 mM, 2 mM, 4 mM, and 8 mM, respectively. Control wells (BC) were set up using the culture medium of Formula 6.

[0096] When using the culture medium of Formula 7, 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 7.

[0097] When using the culture medium of Formula 8, 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 20 ng / mL, 40 ng / mL, 80 ng / mL, 160 ng / mL, and 320 ng / mL, respectively. Control wells (BC) were set up using the culture medium of Formula 8.

[0098] 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 obtained after one generation of culture using each medium to the number of cells obtained after one generation of culture using the corresponding control well. A ratio greater than 1 indicates that the culture medium containing the different concentrations of factors is more effective in promoting cell proliferation than the culture medium in the control well; a ratio less than 1 indicates that the culture medium containing the different concentrations of factors is less effective in promoting cell proliferation than the culture medium in the control well.

[0099] according to Figures 2A to 2HThe results show that human IL-7, human IL-2, human SCF, non-essential amino acids, human TPO, glutamine additives, human IL-3, human FLT3L and other factors have a significant proliferative effect on human primary acute lymphoblastic leukemia cells. According to the results of this embodiment, the content of human IL-7 is preferably 1ng / ml~81ng / ml, more preferably 9ng / ml~27ng / ml, and the concentration of 9ng / mL is the most obvious when added to cell proliferation; the content of human IL-2 is preferably 1ng / ml~81ng / ml, more preferably 3ng / mL~81ng / mL, and the concentration of 27ng / mL is the most obvious when added to cell proliferation; the content of human SCF is preferably 1ng / ml~81ng / ml, more preferably 9ng / mL~81ng / mL, and the concentration of 27ng / mL is the most obvious when added to cell proliferation; the content of non-essential amino acids is preferably 12.5μM~200μM, more preferably 12.5μM~50μM, and the concentration of 25μM is added to cells. The cell proliferation effect is most obvious; the content of human TPO is preferably 1ng / ml~81ng / ml, more preferably 1ng / ml~9ng / mL, and the cell proliferation effect is most obvious when the concentration is 3ng / mL; the content of glutamine additive is preferably 1mM~2mM, and the cell proliferation effect is most obvious when the concentration is 1mM; the content of human IL-3 is preferably 1.89ng / ml~153ng / ml, more preferably 5.67ng / mL~153ng / mL, and the cell proliferation effect is most obvious when the concentration is 51ng / mL; the content of human FLT3L is preferably 20ng / ml~320ng / ml, more preferably 80ng / mL~320ng / mL, and the cell proliferation effect is most obvious when the concentration is 80ng / mL.

[0100] In the following examples, a culture medium containing basal medium, 9 ng / mL human IL-7, 27 ng / mL human IL-2, 27 ng / mL human SCF, 25 μM non-essential amino acids, 3 ng / mL human TPO, 1 mM glutamine supplement, 51 ng / mL human IL-3, and 80 ng / mL human FLT3L was used as the culture medium of the present invention.

[0101] Example 4 Culture and identification of primary human acute lymphoblastic leukemia cells

[0102] (1) Culture of primary human acute lymphoblastic leukemia cells

[0103] According to the method of step (2) of step 3 of Example 1, human primary acute lymphoblastic leukemia cells were obtained from a human primary acute lymphoblastic leukemia bone marrow sample (numbered A10091), and cultured using the human primary acute lymphoblastic leukemia cell culture medium of the present invention (composed of the optimal components and concentration combination determined in Example 3). The obtained human primary acute lymphoblastic 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 lymphoblastic 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.

[0104] The cultured human primary acute lymphoblastic 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 that the number of viable cells had increased 3.73-fold after day 7 of culture.

[0105] (2) Flow cytometry identification of primary human acute lymphoblastic leukemia cells

[0106] According to the method of step (2) of Example 1, primary human acute lymphoblastic leukemia cells were obtained from primary human acute lymphoblastic leukemia bone marrow samples (numbered A09173, A23156, and A29061), and cultured using the human primary acute lymphoblastic leukemia cell culture medium of the present invention. Specifically, the obtained primary human acute lymphoblastic 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.

[0107] Human primary acute lymphoblastic 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 with leukocytes (APC Mouse Anti-Human CD45 (purchased from BD, 560973)) and lymphoid markers (PE Mouse Anti-Human CD20 (purchased from BD, 555623)) 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, the above-mentioned antibodies were added at a ratio of 1:40 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 lymphoid markers in primary human acute lymphoblastic leukemia cells before culture and after 7 days of culture was analyzed using a flow cytometer (Beckman EVOS M500).

[0108] Figures 4A to 4C They are the results of flow cytometry identification of human primary acute lymphoblastic leukemia cells cultured using the human primary acute lymphoblastic leukemia cell culture medium of the present invention. Figures 4A to 4C It was confirmed that after 7 days of continuous culture of primary human acute lymphoblastic leukemia cells cultured in the culture medium of the present invention, the proportion of lymphoid leukemia cells increased by 29.46-57.54%.

[0109] Example 5 Primary Culture Cycle and Cell Number Statistics of Human Primary Acute Lymphoblastic Leukemia Cells and Calculation of Population Doubling (PD) Values

[0110] According to the method of step (2) 3 of Example 1, human primary acute lymphoblastic leukemia cells were obtained from 6 human primary acute lymphoblastic leukemia cell bone marrow samples (numbered A23156, A10097, A23093, A29095, A17076, and A17122). The obtained human primary acute lymphoblastic leukemia cells were cultured at a viable cell density of 1×10 6Cells were seeded in 12-well plates at 400 nm / 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, and each generation was passaged and the corresponding culture cycle was 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.

[0111] Figure 5 The growth curves of six human primary acute lymphoblastic leukemia cells cultured using the human primary acute lymphoblastic leukemia cell culture medium 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 represents 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 cycle, that is, the more cells are expanded. The slope represents the rate of cell expansion. Figure 5 It can be confirmed that when the primary human acute lymphoblastic leukemia cells cultured in the culture medium of the present invention are continuously cultured and expanded for at least 40 days, the cell expansion rate remains basically unchanged and still has the ability to continue to expand.

[0112] Comparison of Example 6 with existing culture medium culture effects

[0113] (1) Preparation of control culture medium

[0114] A control culture medium (Silvia Ravera et al., Scientific Reports, (2020) 10: 16519) was prepared, the formula of which was 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.

[0115] (2) Acquisition and culture of primary human acute lymphoblastic leukemia cells

[0116] Human primary acute lymphoblastic leukemia cells were obtained from a primary human acute lymphoblastic leukemia bone marrow sample (A10091) 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.

[0117] 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 tube was 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 mixed thoroughly with 12 μL of trypan blue dye (Sangon Biotech (Shanghai) Co., Ltd.). 20 μL was 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 counting results are shown in Figure 6 .

[0118] according to Figure 6 The results show that, compared with the control culture medium, the human primary acute lymphoblastic leukemia culture medium of the present invention can significantly promote the expansion of human primary acute lymphoblastic leukemia cells, and its effect is better than the culture medium used in the prior art.

[0119] Example 7: Human primary acute lymphoblastic leukemia cells amplified using the culture medium of the present invention are used for drug screening

[0120] 1. Cell Culture and Plating

[0121] Human primary acute lymphoblastic leukemia cells (numbered A17025) were isolated from the obtained human primary acute lymphoblastic leukemia cells according to the same method as in Example 1, and used as the first generation. The cells were cultured using the human primary acute lymphoblastic leukemia cell culture medium of the present invention, and the cells were passaged after the cells expanded to 85%. 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 lymphoblastic 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.

[0122] 2. Screening drug preparation

[0123] 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.

[0124] Table 3 Drug action concentration settings

[0125]

[0126] 3. High-throughput dosing

[0127] 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 lymphoblastic leukemia cells. After drug addition, the 384-well plate was surface-disinfected and moved to an incubator. Cell viability was measured 72 hours later.

[0128] 4. Cell activity test

[0129] 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).

[0130] 5. Data processing

[0131] 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 .

[0132] Depend on Figures 7A-7F Can confirm, use the people's primary acute lymphoblastic leukemia cell culture medium of the present invention to obtain to carry out drug screening, the same drug is basically consistent for the inhibitory effect of the different generations of cells cultivated (inhibition curve is basically consistent). The sensitivity of the cell of the same patient to different drugs during the maximum blood drug concentration in the human body is different. According to the result, can judge the effectiveness of people's primary acute lymphoblastic leukemia patient when using this kind of medicine clinically, can illustrate that the tumor cell of the different generations that the culture method of the present invention obtains is stable to the sensitivity of drug simultaneously.

[0133] Industrial Applicability

[0134] The present invention provides a primary cell culture medium and a culture method for culturing primary human acute lymphoblastic 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.

[0135] 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 lymphoblastic leukemia cells, characterized in that: Made with the following ingredients: Glutamine supplement; non-essential amino acids; recombinant human FLT3 Ligand; human interleukin-3; human interleukin-7; human interleukin-2; human stem cell factor; human thrombopoietin; an initial culture medium selected from monocyte serum-free medium or RPMI-1640; fetal bovine serum; and an antibiotic selected from one or more of streptomycin / penicillin, amphotericin B, and primocin. in: (1) 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; (2) The content of the glutamine additive in the culture medium is 1 mM to 2 mM; (3) The content of the recombinant human FLT3 Ligand in the culture medium is 20 ng / ml to 320 ng / ml; (4) The content of human interleukin-3 in the culture medium is 1.89 ng / ml to 153 ng / ml; (5) The content of human interleukin-7 in the culture medium is 1 ng / ml to 81 ng / ml; (6) The content of human interleukin-2 in the culture medium is 1 ng / ml to 81 ng / ml; (7) The content of the human stem cell factor in the culture medium is 1 ng / ml to 81 ng / ml; (8) The content of human thrombopoietin in the culture medium is 1 ng / ml to 81 ng / ml.

2. The human primary acute lymphoblastic leukemia cell culture medium according to claim 1, wherein The culture medium of the human primary acute lymphoblastic leukemia cells satisfies any one, multiple or all of the following: (1) The content of the non-essential amino acids in the culture medium is 12.5 µM to 50 µM; (2) The content of the recombinant human FLT3 Ligand in the culture medium is 80 ng / mL to 320 ng / mL; (3) The content of human interleukin-3 in the culture medium is 5.67 ng / mL to 153 ng / mL; (4) The content of human interleukin-7 in the culture medium is 3 ng / ml to 81 ng / ml; (5) The content of human interleukin-2 in the culture medium is 9 ng / mL to 81 ng / mL; (6) The content of the human stem cell factor in the culture medium is 9 ng / mL to 81 ng / mL; (7) The content of human thrombopoietin in the culture medium is 1 ng / ml to 9 ng / mL.

3. A method for culturing primary human acute lymphoblastic leukemia cells, characterized in that: The human primary acute lymphoblastic leukemia cells are cultured using the human primary acute lymphoblastic leukemia cell culture medium according to any one of claims 1 to 2.

4. A drug screening method for human primary acute lymphoblastic leukemia, comprising the following steps: (1) Cultivating primary human acute lymphoblastic leukemia cells using the method for culturing primary human acute lymphoblastic leukemia cells as described in claim 3; (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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