RU2843301C2 - Culture medium and culturing method for primary human cells of acute myeloid leukaemia - Google Patents

Abstract

FIELD: biotechnology.

SUBSTANCE: disclosed are a culture medium and a culturing method for primary human cells of acute myeloid leukaemia. Acute myeloid leukaemia primary human cell culture medium contains glutamine in the form of an additive, non-essential amino acid(s), human interleukin-6, human interleukin-7, human interleukin-3, recombinant human FLT3 ligand, recombinant human macrophage colony-stimulating factor and human stem cell factor. Method for screening drugs for treating human primary acute myeloid leukaemia and a method for evaluating the effectiveness of drugs for treating human primary acute myeloid leukaemia. Invention can be used for culturing primary human cells of acute myeloid leukaemia, evaluating the therapeutic effect and screening medicinal agents.

EFFECT: using said culture medium and method for culturing acute myeloid leukaemia cells can be cultured with higher amplification efficiency and longer cultivation time in vitro.

4 cl, 7 dwg, 3 tbl, 1 ex

Description

Field of technology

The invention relates to the field of cell culturing technology, in particular to a culture medium for primary cells for culturing primary human acute myeloid leukemia (AML) cells in vitro, a method for using said culture medium for culturing primary human acute myeloid leukemia cells and their use for assessing the effectiveness and screening of drugs.

State of the art

Acute myeloid leukemia (AML) is a clonal malignant proliferative disorder of the blood with the presence of myeloid blasts in the hematopoietic system. AML is an aggressive disease characterized by high genetic diversity and the presence of biologically and prognostically distinct subtypes. AML affects 1 to 5 people per 100,000 population each year and accounts for 30-40% of all new cases of leukemia. AML is the most lethal leukocyte disorder with the worst prognosis and survival (<26% at 5 years), which have 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).

AML stem cells are currently the main focus of leukemia research because they have acquired resistance and mediate the formation of relapsed leukemic cell subclones. Research work usually requires culturing AML stem cells. Culturing AML stem cells is challenging due to the heterogeneity in the AML stem cell population and the fact that many patients have poorly grown cells that are prone to differentiation and lose their ability to self-renew in vitro. In addition, primary cells from AML patients can be difficult to obtain and, unlike transformed cell lines, they cannot be expanded indefinitely in vitro. In vivo xenograft models allow expansion of human AML cells but require large numbers of cells (>10 ⁶ cells/mouse) and do not provide maintenance for AML cells. On the other hand, co-culture with bone marrow (BM)-derived mesenchymal stromal cells (MSCs) or stromal cell lines may help maintain AML stem cells, but feeder cell cultures are not suitable for high-throughput drug screening or adaptation testing.

Thus, there is a need in the art for a culture medium and a culturing method for long-term, rapid expansion of primary human acute myeloid leukemia cells without co-cultivation with stromal cells.

Brief description of the invention

To solve the above technical problems, the present invention proposes a culture medium and a cultivation method for rapid reproduction of primary human acute myeloid leukemia cells in vitro.

One aspect of the invention is to provide a culture medium for primary human acute myeloid leukemia cells, wherein the culture medium comprises glutamine as an additive, 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).

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

(1) the amount of glutamine as an additive in the culture medium is preferably from 0.5 mM to 4 mM;

(2) the non-essential amino acid(s) is(are) one or more amino acids selected from the group consisting of glycine, alanine, asparagine, aspartic acid, thymic acid, proline and serine, and the amount of the non-essential amino acid(s) in the culture medium is preferably from 12.5 μM to 200 μM;

(3) the amount of human IL-6 in the culture medium is preferably from 1.89 ng/ml to 17 ng/ml;

(4) the amount of human IL-7 in the culture medium is preferably from 1.89 ng/ml to 51 ng/ml;

(5) the amount of human IL-3 in the culture medium is preferably from 1.89 ng/ml to 153 ng/ml;

(6) the amount of human FLT3L in the culture medium is preferably from 3 ng/ml to 81 ng/ml;

(7) the amount of human M-CSF in the culture medium is preferably from 1 ng/ml to 81 ng/ml;

(8) The amount of human FSC in the culture medium is preferably from 1 ng/ml to 81 ng/ml.

In another preferred embodiment, the culture medium for primary human acute myeloid leukemia cells according to the present invention further comprises a basal medium which comprises a starting medium selected from the group consisting of monocytic serum-free medium and RPMI-1640, 5-10% (v/v) fetal bovine serum, and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B, and Primocin. Specifically, in the case of using streptomycin/penicillin, the concentration range of streptomycin is 25-400 μg/ml, preferably 50-200 μg/ml, the concentration range of penicillin is 25-400 U/ml, preferably 50-200 U/ml; in the case of using amphotericin B, the concentration range is 0.25-400 μg/ml, preferably 0.5-200 μg/ml; and in the case of using Primocin, the concentration range is 25-400 μg/ml, preferably 50-200 μg/ml.

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

In a preferred embodiment, the method for culturing primary human acute myeloid leukemia cells in vitro according to the present invention comprises the following steps:

1. Isolation and processing of primary human acute myeloid leukemia cells.

(1) Centrifugation of bone marrow samples from patients with acute myeloid leukemia at 1200-1600 rpm for 2-6 minutes.

(2) After centrifugation: discarding the upper layer of blood plasma, adding 1×PBS (phosphate-buffered saline) in an amount 2-3 times the amount of blood cell sediment to the blood cell sediment to dilute and mixing them thoroughly; adding 6-8 ml of human peripheral blood lymphocyte separation medium to the above diluted blood cell sediment, and centrifuging the resulting mixture at a speed of 380-420×g, acceleration of 1-2, deceleration of 0, temperature of 20-28°C, and a centrifugation time of 25-35 minutes.

(3) After centrifugation to form layers in a centrifuge tube, aspirate the lymphocyte layer into 3-6 ml 1×PBS and mix thoroughly to wash the cells, and then centrifugate at 1200-1600 rpm for 2-6 minutes.

(4) Remove supernatant, add red blood cell lysis buffer to resuspend cell pellet and lyse for 15-20 minutes, centrifuge at 1200-1600 rpm for 2-6 minutes.

(5) Removing the supernatant after centrifugation and adding the basic medium for subsequent use.

2. Cultivating said cells using a culture medium for primary human acute myeloid leukemia cells according to the present invention.

Resuspending the primary human acute myeloid leukemia cells obtained in step 1 above in a culture medium for primary human acute myeloid leukemia cells according to the present invention and counting the cells, inoculating the cells into a culture dish at a density of 1×10 ⁵ to 4×10 ⁶ cells/cm ² and passaging the cells when the cells increase to cover 90% of the culture dish.

In another aspect, the cells obtained by the culturing method according to the present invention can be used in regenerative medicine, basic medical research of acute myeloid leukemia cells, drug response screening and development of new drugs for acute myeloid leukemia. Therefore, the invention also relates to a method for screening or evaluating the effectiveness of drugs for the treatment of primary human acute myeloid leukemia, comprising the steps of:

(1) culturing primary human acute myeloid leukemia cells by the method for culturing primary human acute myeloid leukemia cells according to the present invention;

(2) selection of the drug to be tested and dilution of the drug to obtain different concentration gradients;

(3) adding the diluted drug to the cells cultured and obtained in step (1) and determining the viability of the cells.

The technical solution according to the present invention allows achieving the following technical effects:

(1) the success rate of primary human acute myeloid leukemia cell culture is improved, achieving a success rate of 80% or more;

(2) ensures that primary human acute myeloid leukemia cells cultured in vitro retain the pathological characteristics of patients;

(3) the primary human acute myeloid leukemia cells are highly efficient in propagating, successfully propagating to a number of ¹⁰⁶ primary human acute myeloid leukemia cells within approximately one week from an initial number of ¹⁰⁵ cells, and the propagated primary human acute myeloid leukemia cells have the ability to be continuously passaged;

(5) the cost of cultivation is controllable since the culture medium does not require expensive factors such as Wnt agonists, R-spondin family proteins, BMP inhibitors, FGF10;

(6) This technology enables the cultivation and provision of primary human acute myeloid leukemia cells in large quantities and with high homogeneity, which is suitable for high-throughput screening of new candidate compounds and high-throughput functional in vitro drug susceptibility tests for patients.

Description of drawings

Fig. 1 is a graph showing the effect of different combinations of added factors on the proliferation of primary human acute myeloid leukemia cells.

Figures 2A-2H are graphs showing the effect of different concentrations of each added factor on the proliferation of primary human acute myeloid leukemia cells.

Fig. 3 is a microscopic photograph of primary human acute myeloid leukemia cells cultured using a culture medium for primary human acute myeloid leukemia cells according to the present invention.

Fig. 4 is an image showing the results of identification by flow cytometry of primary human acute myeloid leukemia cells cultured using a culture medium for primary human acute myeloid leukemia cells according to the present invention.

Fig. 5 shows a growth curve of primary human acute myeloid leukemia cells cultured in vitro using the culture medium for primary human acute myeloid leukemia cells according to the present invention.

Fig. 6 is a graph showing a comparison of culturing primary human acute myeloid leukemia cells using a culture medium for primary human acute myeloid leukemia cells according to the present invention and a culture medium of the prior art.

Figs. 7A-7F show dose-response curves for different passages of primary human acute myeloid leukemia cells cultured using the culture medium for primary human acute myeloid leukemia cells according to the present invention for various drugs.

Detailed description of the invention

In order to better understand the present invention, it is further described below together with examples and drawings. The following examples are given solely for the purpose of illustrating the present invention, but not for the purpose of defining it.

Example 1. Effect of appropriate added factors in the culture medium for primary human acute myeloid leukemia cells on the proliferation of primary human acute myeloid leukemia cells

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

First, the basal medium was prepared. The composition of the basal medium was monocytic serum-free medium (purchased from BI, 05-080-1 A) + 10% (v/v) fetal bovine serum (purchased from ExCell Bio, FND500) + 100 μg/ml Primocin (purchased from InvivoGen, 0.2% (v/v), the concentration of the commercial product is 50 mg/ml).

Different types of growth factors (see Table 1) were added to the basal medium to obtain culture media for primary human acute myeloid leukemia cells containing various additional components.

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

1. Selecting a sample

Bone marrow samples were collected from AML patients by professional medical staff in a professional medical institution, and all patients signed informed consent forms. Bone marrow samples of 3 to 10 mL were stored in tubes with EDTA-K.2 anticoagulant (manufacturer: Jiangsu Rongye) and transported under refrigeration at 4-8°C.

2. Preparation of materials

After surface sterilization, sterile 15 ml centrifuge tubes, dispensers, 10 ml pipettes and sterile pipette tips were placed in an ultra-clean workspace and exposed to UV light for 30 min. 1×PBS was removed from a 4°C refrigerator 30 min beforehand.

3. Selecting a sample

3.1 In an ultra-clean workstation, bone marrow samples were mixed by pipetting up and down, transferred to a 15 ml centrifuge tube and centrifuged at 1500 rpm for 4 minutes at room temperature;

3.2 6 ml of human peripheral blood lymphocyte separation medium (purchased from Solarbio, P8610) was added to a new 15 ml centrifuge tube; after centrifuging the bone marrow sample, the upper plasma layer of the blood was discarded, and 1× PBS was added to the blood cell pellet in an amount of 2-3 times the amount of the blood cell pellet for dilution and mixed thoroughly; the diluted blood was slowly layered on the surface of the separation medium layer on the wall of the centrifuge tube, paying attention to keeping the liquid surface clean; the centrifuge tube containing the bone marrow sample mixture was carefully placed into the centrifuge and centrifuged at 400×g for 30 minutes (acceleration: 2, deceleration: 0, temperature: 25°C);

3.3 After centrifugation, the cells in the centrifuge tube were separated into four layers (from top to bottom: PBS layer, milky white ring-shaped lymphocyte layer, separation medium layer, and red blood cell layer); the lymphocyte layer was pipetted into a 15 ml centrifuge tube with 5 ml of 1 x PBS added in advance; the resulting mixture was gently mixed to wash the cells and centrifuged at 1500 rpm for 5 minutes at room temperature;

3.4 The supernatant was discarded and the resulting pellet was examined to determine the presence of blood cells; if blood cells were present, 8 ml of red blood cell lysis buffer (purchased from Sigma, R7757-100ML) was added, then mixed well, lysed at 4°C for 20 minutes with one inversion mixing during the process; the resulting mixture was centrifuged at 1500 rpm at room temperature for 4 minutes;

3.5 The supernatant was discarded and 2 ml of the main medium was added to the resulting sediment to resuspend the cells for subsequent use.

4 Cell counting and processing

4.1 Viable cell count: 12 μl of the resuspended cell suspension was completely 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 slide (Countstar, package: 50 pcs/box); the percentage of viable large cells (cell size>10 μm) was calculated using a cell counter (Countstar, IC1000) (the percentage of viable large cells (cell size>10 μm) was calculated).

cells>10 µm) = number of viable cells/total number of cells×100%).

(3) Cultivation of primary human acute myeloid leukemia cells

The culture media with different compositions shown in Table 1 were added to a 96-well plate at a volume of 100 μl/well. Primary human acute myeloid leukemia cells isolated from bone marrow samples of two cases of primary human acute myeloid leukemia (numbered as A 17007, A2 5104, from the First Affiliated Hospital of Anhui Medical University) according to the above step (2) were inoculated into the 96-well culture plate at a density of 1× ¹⁰⁴ cells/well and cultured at 37°C and 5% _CO2 . After 5-8 days of culture, the cells grew to 70-85%, and 10 μl of cell counting kit 8 reagent (CCK-8, purchased from MCE) was added to each well and incubated at 37°C and 5% _CO2 for 2-4 hours. The substances in each well were mixed, and the plate was read using a multi-function microplate reader (Multi-Mode Detection Platform, American Molecular Instruments (Shanghai) Co., Ltd.) at 450 nm. The basic medium without adding any supplement was used as a control. The experimental results are shown in Table 1.

In the above table, "+" indicates that, compared with the basic medium, the culture medium with the supplement can stimulate the proliferation of primary human cells from two cases of acute myeloid leukemia isolated from bone marrow samples of people with primary acute myeloid leukemia; "-" indicates that the culture medium with the supplement can stimulate the proliferation of primary human cells from at least one case of acute myeloid leukemia isolated from bone marrow samples of people with primary acute myeloid leukemia; "o" indicates that the medium with the supplement has no significant effect on the proliferation of primary human cells from at least two cases of acute myeloid leukemia isolated from bone marrow samples of people with primary acute myeloid leukemia.

Based on the above results, factors such as human IL-7, human IFN-a, 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.

Example 2. Effect of different combinations of added factors in the culture medium for primary human acute myeloid leukemia cells on the proliferation of primary human acute myeloid leukemia cells

Culture media for primary human acute myeloid leukemia cells containing different combinations of added factors were prepared according to the components listed in Table 2 to study the proliferation-promoting effect of different combinations of added factors on primary human acute myeloid leukemia cells.

Primary human acute myeloid leukemia cells were obtained from bone marrow samples from individuals with primary acute myeloid leukemia (numbered A 17151, A 17152, A 20090, A 20101, A 21004,

A 20141) according to the method described in step (2) to 3 of Example 1. The resulting cell suspension was divided into 11 equal parts, which were then centrifuged at 1500 rpm for 4 minutes. After centrifugation, the cells were resuspended in 200 μl of OS or culture medium No. 1-10, respectively, and inoculated into a 48-well plate at a viable cell density of 2 × 10 ⁴ cells/well (20,000 cells per well). The respective culture media were added to each well in the 48-well plate to make a volume of 1 ml, and the resulting suspension was thoroughly mixed. After disinfecting the surface, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C, 5% CO ₂ for culturing.

The cells in the 48-well plate grew to more than 85% and were transferred to a 15-mL centrifuge tube, which was then centrifuged at 1500 rpm for 5 minutes. 500 μL of monocytic 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 then 20 μL of this mixture was added to a cell counting slide (Countstar, packing: 50 pcs. per box). The percentage of viable large cells (cell size>10 μm) was calculated using a cell counter (Countstar, IC1000) (percentage of viable large cells (cell size>10 μm)=number of viable cells/total number of cells×100%). The results obtained for primary human acute myeloid leukemia cells from bone marrow samples A 17151, A 17152, A 20090, A 20101, A 21004, and A 20141 are shown in Fig. 1.

According to the results in Fig. 1, it can be seen that, compared with the basic medium, the use of culture media No. 1-10 can promote the proliferation of primary human acute myeloid leukemia cells to different degrees. The composition of culture medium No. 2 does not contain human IFN-a, which unexpectedly leads to a better effect on proliferation. The results indicate that factors such as glutamine as 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 primary human acute myeloid leukemia cells.

Example 3. Proliferative effect of different concentrations of added factors on primary human acute myeloid leukemia cells

Primary human acute myeloid leukemia cells were obtained from bone marrow samples of humans with primary acute myeloid leukemia (numbered A 23065, A 17112) according to the method described in step (2)-3 of Example 1. The cells were resuspended in basal medium (monocyte serum-free medium+10% (v/v) fetal bovine serum+100 μg/ml Primocin) for subsequent use.

Then, the factors with the effect promoting cell culture proliferation determined in Example 2 were added to the basic medium (prepared according to composition No. 2 of Example 2) to obtain a combined basic medium, and then the following 8 compositions of culture media for experiments were obtained.

Composition 1: combined basic medium without glutamine as an additive;

Composition 2: combined basic medium without human FSC;

Composition 3: combined basic medium without human IL-6;

Composition 4: combined basic medium without human IL-3;

Composition 5: Combined basal medium without human FLT3L;

Composition 6: combined basic medium without non-essential amino acids;

Composition 7: Combined basic medium without human IL-7;

Composition 8: Combined basic medium without human M-CSF.

20 μl of cell suspension containing 4× ¹⁰⁴ cells were added to each well, and the suspension was diluted with 1 ml of the medium of the above-mentioned Compositions 1-8, respectively.

When using the Composition 1 culture medium, the prepared glutamine supplement was added to the 48-well plate with inoculated primary cells at 1 ml per well, respectively, and the final concentration of glutamine in the form of supplement was 0.5 mM, 1 mM, 2 mM, 4 mM, 8 mM, respectively; a blank control (BC) well was prepared using the Composition 1 culture medium.

When using the Composition 2 culture medium, the prepared human FSC was added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of human FSC was 1 ng/ml, 3 ng/ml, 9 ng/ml, 27 ng/ml, 81 ng/ml, respectively; a blank control (BC) well was prepared using the Composition 2 culture medium.

When using the Composition 3 culture medium, the prepared human IL-6 was added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of human IL-6 was 1.89 ng/ml, 5.67 ng/ml, 17 ng/ml, 51 ng/ml, 153 ng/ml, respectively; a blank control (BC) well was prepared using the Composition 3 culture medium.

When using the Composition 4 culture medium, the prepared human IL-3 was added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of human IL-3 was 1.89 ng/ml, 5.67 ng/ml, 17 ng/ml, 51 ng/ml, 153 ng/ml, respectively; a blank control (BC) well was prepared using the Composition 4 culture medium.

When using the Composition 5 culture medium, the prepared human FLT3L was added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of human FLT3L was 1 ng/ml, 3 ng/ml, 9 ng/ml, 27 ng/ml, 81 ng/ml, respectively; a blank control (BC) well was prepared using the Composition 5 culture medium.

When using the Composition 6 culture medium, the prepared non-essential amino acids were added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of non-essential amino acids was 12.5 μM, 25 μM, 50 μM, 100 μM, 200 μM, respectively; a blank control (BC) well was prepared using the Composition 6 culture medium.

When using Composition 7 culture medium, the prepared IL-7 was added to the 48-well plate with inoculated primary cells at 1 ml per well, respectively, and the final concentration of IL-7 was 1.89 ng/ml, 5.67 ng/ml, 17 ng/ml, 51 ng/ml, 153 ng/ml, respectively; a blank control (BC) well was prepared using Composition 7 culture medium.

When using Composition 8 culture medium, the prepared human M-CSF was added to the 48-well plate inoculated with primary cells at 1 ml per well, respectively, and the final concentration of human M-CSF was 1 ng/ml, 3 ng/ml, 9 ng/ml, 27 ng/ml, 81 ng/ml, respectively; a blank control (BC) well was prepared using Composition 8 culture medium.

When the cells expanded to cover approximately 85% of the area of 48 wells, the proliferation ratio relative to the number of cells in the blank control well (BC) was calculated, and the results were shown in Figs. 2A to 2H, respectively. In Figs. 2A to 2H, the ratio represents the ratio of the number of first-passage cells cultured using each culture medium to the number of first-passage cells cultured in the corresponding blank control well. If the ratio is greater than 1, it indicates that the proliferation-promoting effect of the resulting culture medium containing different concentrations of factors or low-molecular compounds is preferable to that of the culture medium in the blank control well; if the ratio is less than 1, it indicates that the proliferation-promoting effect of the resulting culture medium containing different concentrations of factors or low-molecular compounds is weaker than that of the culture medium in the blank control well.

According to the results in Figs. 2A to 2H, glutamine as a supplement, human CSF, human IL-6, human IL-3, human FLT3L, non-essential amino acids, human IL-7, and human M-CSF exert significant proliferation-promoting effects on primary human acute myeloid leukemia cells. According to the results of this Example, the amount of glutamine as a supplement is preferably 0.5 mM to 4 mM, and the effect on cell proliferation is most significant when added at a concentration of 0.5 mM; the amount of human CSF is preferably 1 ng/mL to 81 ng/mL, and the effect on cell proliferation is most significant when added at a concentration of 9 ng/mL; the amount of human IL-6 is preferably from 1.89 ng/ml to 17 ng/ml, and the effect on cell proliferation is most significant when added at a concentration of 5.67 ng/ml; the amount of human IL-3 is preferably from 1.89 ng/ml to 153 ng/ml, and the effect on cell proliferation is most significant when added at a concentration of 51 ng/ml; the amount of human FLT3L is preferably from 3 ng/ml to 81 ng/ml, and the effect on cell proliferation is most significant when added at a concentration of 27 ng/ml; the amount of non-essential amino acids is preferably from 12.5 μM to 200 μM, and the effect on cell proliferation is most significant when added at a concentration of 50 μM; the amount of human IL-7 is preferably from 1.89 ng/ml to 51 ng/ml, and the effect on cell proliferation is most significant when added at a concentration of 17 ng/ml; the amount of human M-CSF is preferably from 1 ng/ml to 81 ng/ml, and the effect on cell proliferation is most significant when added at a concentration of 27 ng/ml.

Example 4. Cultivation and identification of primary human acute myeloid leukemia cells.

(1) Cultivation of primary human acute myeloid leukemia cells

Primary human acute myeloid leukemia cells were obtained from bone marrow samples of humans with primary acute myeloid leukemia (numbered A17030) according to the method described in step (2) to 3 of Example 1, and cultured using a culture medium for primary human acute myeloid leukemia cells according to the invention (the composition is the optimal combination of components and concentration determined in Example 3, i.e., contains a basic medium, 0.5 mM glutamine as a supplement, 9 ng/ml human CSF, 5.67 ng/ml human IL-6, 51 ng/ml human IL-3, 27 ng/ml human FLT3L, 50 μM of essential amino acids, 17 ng/ml human IL-7 and 27 ng/ml human M-CSF). The obtained primary human acute myeloid leukemia cells were inoculated into a 6-well plate at a viable cell density of 3 x 10 ⁶ cells/well. 5 ml of the culture medium for primary human acute myeloid leukemia cells according to the present invention was added to the plate, and the resulting suspension was mixed well. After disinfecting the surface, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C, 5% CO ₂ for culturing.

The cultured primary human acute myeloid leukemia cells were examined under a microscope (EVOS M500, Invitrogen). Fig. 3 shows photographs after 1 day, 4 days, and 7 days of culture taken with a 10x magnification eyepiece. According to the cell counting results, the multiplicity of increase in the number of viable cells after 7 days of culture was up to 3.58 times.

(2) Identification of primary human acute myeloid leukemia cells by flow cytometry

Primary human acute myeloid leukemia cells were obtained from bone marrow samples of humans with primary acute myeloid leukemia (numbered as A 17014) according to the method described in Step (2) to 3 of Example 1, and cultured using a culture medium for primary human acute myeloid leukemia cells according to the present invention. Specifically, the obtained primary human acute myeloid leukemia cells were inoculated into a 12-well plate at a viable cell density of 1×10 ⁶ cells/well. 3 ml of the culture medium according to the invention were added to the plate, and the resulting suspension was mixed well. After disinfecting the surface, the plate was placed in an incubator (purchased from Thermo Fisher) at 37°C, 5% CO ₂ for culture.

Primary human acute myeloid leukemia cells were transferred to 15 ml centrifuge tubes before and after 7 days of culture, respectively, and centrifuged at 1500 rpm for 5 minutes at room temperature. The supernatant was discarded, the cell pellet was diluted with 2 ml of 1×PBS and equally divided into 2 parts (one part served as an experimental group for double labeling with a leukocyte marker (APC mouse anti-human CD45 antibody (purchased from BD, 560973)) and a myeloid marker (BB 515 mouse anti-human CD33 antibody (purchased from BD, 564588)); the other part served as a control group) in 1.5 ml centrifuge tubes, which were then centrifuged at room temperature for 5 min at 1500 rpm. The supernatant was discarded and 40 μl of 0.5% BSA (prepared in 1×PBS) was added to the centrifuge tube. Then, the above antibodies were added to the cells at a dilution of 1:40 in the dark and mixed well. No antibodies were added to the control group. The cells were incubated on ice for 1-2 hours. After incubation, 1 ml of 1×PBS was added to each tube for resuspension and washing, and centrifuged at room temperature at 1500 rpm for 5 minutes. The supernatant was discarded, 300 μl of 1×PBS was added to resuspend the cell pellet; flow cytometry (Beckman EVOS M500) was used to analyze the expression of myeloid marker in primary human acute myeloid leukemia cells before culturing and after culturing for 7 days, respectively.

Fig. 4 shows the result of identification by flow cytometry of primary human acute myeloid leukemia cells cultured using the culture medium for primary human acute myeloid leukemia cells according to the present invention. The data in Fig. 4 confirm that after 7 days of continuous culture of primary human acute myeloid leukemia cells in the medium according to the invention, the proportion of myeloid leukemia cells increased by 16%.

Example 5. Statistics of the first culture period and the number of primary human acute myeloid leukemia cells and calculation of the population doubling (PD) value.

According to the method described in Step (2) to 3 of Example 1, primary human acute myeloid leukemia cells were obtained from 8 bone marrow cell samples of humans with primary acute myeloid leukemia (numbered A 23123, A 15094, A 23133, A 23123-2, A 23023, A 14003, A 23124, A 09169). The obtained primary human acute myeloid leukemia cells were inoculated into a 12-well plate at a viable cell density of 1 x 10 ⁶ cells/well 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 until the time of passage was regarded as a culture cycle. Under these experimental conditions, the expanded cells were expanded in further passages. After each passage, the cells were counted and the corresponding culture cycle was recorded. The PD value was calculated using the formula: population doubling (PD) = 3.32 × 1d (total number of cells after detachment / initial number of inoculated cells). For the formula, see Chapman et al., Stem Cell Research & Therapy 2014, 5: 60.

Fig. 5 shows the growth curves for 8 cases of primary cells cultured using the culture medium for primary human acute myeloid leukemia cells according to the present invention, plotted using the Graphpad Prism software. The abscissa axis represents the days of cell culture, and the ordinate axis represents the cumulative cell proliferation rate, i.e., the cell multiplication rate in a culture cycle. The larger the value, the more times the cells multiplied in a certain cycle, i.e., the more cells multiply. The slope represents the cell multiplication rate. The data in Fig. 5 confirm that when the primary human acute myeloid leukemia cells cultured in the culture medium according to the present invention were continuously cultured and multiplied for at least 45 days, the cell multiplication rate remained basically unchanged, and the cells still had the ability to continue to multiply.

Example 6. Comparison of the effect on cultivation with the culture medium of the prior art

(1) Obtaining control culture medium

The control culture medium of the prior art (Silvia Ravera et al., Scientific Reports, (2020) 10:16519) was prepared with the composition: 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% fetal bovine serum (FBS, purchased from ExCell Bio, FND500) (hereinafter referred to as “control culture medium”).

(2) Obtaining and culturing primary human acute myeloid leukemia cells

Primary human acute myeloid leukemia cells were obtained from a bone marrow sample of a human with primary acute myeloid leukemia (A 10093) according to the method described in step (2)-3 of Example 1, inoculated into a 12-well plate at a viable cell density of 1×10 ⁶ cells/well, and cultured in a culture medium according to the invention and a control culture medium, respectively.

On the 7th day of culture, the 12-well plate was removed. The cell culture was transferred to a 15-mL centrifuge tube, centrifuged at 1500 rpm for 5 min at room temperature, and the cell pellet was resuspended in 1 mL of the 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 then 20 μL of this mixture was added to a cell counting slide (Countstar, package: 50 pcs. per box). The total number of cells was counted using a cell counter (Countstar, IC1000). The counting results are shown in Fig. 6.

According to the results shown in Fig. 6, it is seen that, compared with the control culture medium, the culture medium for primary human acute myeloid leukemia cells according to the present invention can significantly promote the proliferation of primary human acute myeloid leukemia cells, and its effect is better than that of the control culture medium.

Example 7. Use of primary human acute myeloid leukemia cells expanded using the culture medium according to the present invention for drug screening and efficacy evaluation

1. Cell culturing and seeding

According to the same method as in Example 1, primary human acute myeloid leukemia cells (numbered A 23170) were isolated and used for expansion, cultured using a culture medium for primary human acute myeloid leukemia cells according to the present invention until the cells were expanded to 85% coverage, and then passaged. The cells were passaged and counted according to the steps in Example 1.

The cells were loaded into a loading slot (purchased from Corning) at a viable cell density of 1× ¹⁰⁵ cells/mL and mixed thoroughly. They were then loaded into a 384-well opaque white cell culture plate (purchased from Corning) at a volume of 50 μL per well and a cell count of 5000 cells/well. The plate was filled to the brim with primary human acute myeloid leukemia cell culture medium according to the invention, and the sample names, dosing ratios, and CellTiter-Glo assay numbers (Promega) were noted on the plate. The plate surface was disinfected with 75% alcohol (LIRCON), the plate was cultured in an incubator at 37°C, 5% _CO2 , and doses were administered after 24 hours. For drug screening, cells of the 1st, 2nd, 3rd, 4th and 5th culture passages were obtained, respectively, and the drug sensitivity of primary cells cultured using the culture medium according to the present invention for continuous passaging was tested.

2. Preparation of drug candidates.

Six drugs (cytarabine, doxorubicin, bortezomib, panobinostat, azacitidine, and homoharringtonine; all purchased from MCE) were prepared in 6 concentration gradients according to the following table and added to a 384-well plate (Thermo Fisher) at a volume of 30 μl per well and stored until use.

3. High-performance dosing

The prepared drug plate was removed and kept at room temperature. The plate was centrifuged at room temperature for 1 minute at 1000 rpm in a centrifuge (Beckman) and then removed. For high-throughput dosing, a high-throughput automated workstation (JANUS, Perkin Elmer) was used. 0.1 μl of drug candidates at appropriate concentrations were added to each well of a 384-well plate with cultured primary human acute myeloid leukemia cells. After dosing, the surface of the 384-well plate was disinfected and placed in an incubator. Cell viability was measured after 72 hours.

4. Measuring cell viability

CellTiter-Glo Fluorescent Reagent (Promega) was removed from the 4°C refrigerator and 10 ml of reagent was added to the loading slot. The 384-well assay plate was removed from the incubator and 10 µl of CellTiter-Glo Fluorescent Reagent was added to each well. After a 10-minute incubation, the assay was run using a multi-plate reader (Envision, 20 Perkin Elmer).

5. Data processing

According to the formula: cell inhibition rate (%)=100%-chemiluminescence value of drug-dosed well/chemiluminescence value of control well×100%, 25 the inhibition rate of cells treated with different drugs was calculated, and the half-inhibition concentration (IC ₅₀ ) of cells for the drugs was calculated using Graphpad Prism software. The results are shown in Figs. 7A to 7F.

The data in Fig. 7A-7F confirm that when used for screening

of the medicinal products of primary human acute myeloid leukemia cells cultured in the culture medium for primary human acute myeloid leukemia cells according to the present invention, the inhibitory effect of the same medicinal product on the cultured cells of different passages remains substantially unchanged (the inhibition curves are substantially similar). Cells from the same patient have different sensitivities to different medicinal products at the maximum concentration in the blood of the human body. Based on the results obtained, it is possible to judge the efficacy of the medicinal product in clinical use in patients with primary human acute myeloid leukemia. At the same time, the results indicate that the sensitivity of the tumor cells of different passages obtained in accordance with the culturing method according to the invention to medicinal products is stable.

Industrial applicability

The present invention provides a culture medium for primary cells and a culture method for culturing primary human acute myeloid leukemia cells in vitro, and the cultured cells can be used for evaluating the efficacy and screening of drugs. Thus, the present invention is suitable for industrial application.

Although the invention has been described in detail in the present application, the present invention is not limited thereto. Those skilled in the art can make modifications in accordance with the principles of the present invention. Therefore, it should be understood that any modifications made in accordance with the principles of the present invention are within the scope of protection of the present invention.

Claims (20)

1. Culture medium for culturing primary human acute myeloid leukemia cells, containing: glutamine as a supplement, a non-essential amino acid(s), human interleukin-6, human interleukin-7, human interleukin-3, recombinant human FLT3 ligand, recombinant human macrophage colony-stimulating factor, human stem cell factor, a basal medium that comprises a base medium selected from the group consisting of monocytic serum-free medium and RPMI-1640, fetal bovine serum, and one or more antibiotics selected from the group consisting of streptomycin/penicillin, amphotericin B, and Primocin, wherein (1) the amount of glutamine added to the culture medium is from 0.5 mM to 4 mM; (2) the non-essential amino acid(s) is/are one or more amino acids 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(s) in the culture medium is from 12.5 μM to 200 μM; (3) the amount of human interleukin-6 in the culture medium ranges from 1.89 ng/ml to 17 ng/ml; (4) the amount of human interleukin-7 in the culture medium ranges from 1.89 ng/ml to 51 ng/ml; (5) the amount of human interleukin-3 in the culture medium ranges from 1.89 ng/ml to 153 ng/ml; (6) the amount of recombinant human FLT3 ligand in the culture medium ranges from 3 ng/ml to 81 ng/ml; (7) the amount of recombinant human macrophage colony-stimulating factor in the culture medium ranges from 1 ng/ml to 81 ng/ml; (8) The amount of human stem cell factor in the culture medium ranges from 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 cultivated using a culture medium for primary human acute myeloid leukemia cells according to paragraph 1. 3. A method for screening drugs for the treatment of human primary acute myeloid leukemia, comprising the following steps: (1) culturing primary human acute myeloid leukemia cells by the method for culturing primary human acute myeloid leukemia cells according to claim 2; (2) selection of the drug to be tested and dilution of the drug to obtain different concentration gradients; (3) adding the diluted drug to the cells cultured and obtained in step (1) and determining the viability of the cells. 4. A method for assessing the efficacy of drugs for the treatment of human primary acute myeloid leukemia, including the following stages: (1) culturing primary human acute myeloid leukemia cells by the method for culturing primary human acute myeloid leukemia cells according to claim 2; (2) selection of the drug to be tested and dilution of the drug to obtain different concentration gradients; (3) adding the diluted drug to the cells cultured and obtained in step (1) and determining the viability of the cells; (4) evaluation of the efficacy of drugs for the treatment of human primary acute myeloid leukemia based on the results obtained in stage (3).