In-vitro cell culture method for primary B cell acute lymphoblastic leukemia

A technology for in vitro culture of leukemia cells, applied in the field of in vitro culture of primary B-cell acute lymphoblastic leukemia cells, can solve the problems of long time-consuming, high cost, and low success rate of culture, and achieve the effect of facilitating promotion and high success rate

Active Publication Date: 2015-04-08
GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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  • Claims
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AI Technical Summary

Problems solved by technology

Although patient-derived B-ALL sample cells can be xenotransplanted into immunodeficient mice to expand and proliferate, and then conduct in vivo experiments, the in vivo research is costly and time-consuming, especially for new therapeutic methods or drug screening experiments. material resources are unpredictable
[0004] Some related reports on the in vitro culture of primary B-ALL cells have been published, such as the use of human cytokines SCF, IL-3, IL-7 and FLT-3L to stimulate culture, or the use of normal human bone marrow endothelial cell line (HBME, human bone marrow endothelial cells) or human bone marrow stromal cells (MSC, human marrow stromal cell), etc., but the success rate of culture is low and the reproducibility of the results is poor
[0005] To sum up, the existing technology using B-ALL cell lines for tumor research cannot truly simulate the heterogeneity of tumors and cannot meet the treatment needs of tumors that mutate with drug use; while the in vitro culture of primary B-ALL cells Still in the immature stage, most tumor cell samples undergo rapid apoptosis (within 2 weeks) due to inability to adapt to changes in the internal and external environment, while the proportion of tumor cell samples that can be successfully cultured in vitro for a long time is not high

Method used

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  • In-vitro cell culture method for primary B cell acute lymphoblastic leukemia
  • In-vitro cell culture method for primary B cell acute lymphoblastic leukemia
  • In-vitro cell culture method for primary B cell acute lymphoblastic leukemia

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Experimental program
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Effect test

Embodiment 1

[0030] 1. Culture of OP9 cell line

[0031] OP9 medium: αMEM medium (HyClone, Thermo Scientific, MA, USA), 20% fetal bovine serum (Gibco, Life Technologies, NY, USA), 2mM L-glutamine, 100U / ml penicillin, and 100ug / ml streptomycin.

[0032] Cell passage (adhesion):

[0033] 1) Cells are cultured and maintained in a T75 culture dish. First, use a Pasteur pipette to suck out the culture medium, and wash it once with 2ml PBS;

[0034] 2) Add 2.0-3.0mL 0.25% (w / v) trypsin-0.53mM EDTA solution and incubate for 5-15 minutes until the cell layer is observed to float and disperse under the microscope;

[0035] Note: To avoid cell clumps, do not shake the flask while waiting for the cells to detach. Cells are difficult to separate and can be placed at 37°C to facilitate trypsin catalysis.

[0036] 3) Add 6.0-8.0mL of medium, and gently blow off the cells with a Pasteur pipette;

[0037] 4) Transfer the cell suspension to a 15mL centrifuge tube with a Pasteur pipette, centrifuge at 1...

Embodiment 2

[0046] Embodiment 2: the processing of B-ALL sample

[0047] 1) Mix the bone marrow sample of the B-ALL patient with normal saline in equal proportions;

[0048] 2) In a new 15mL centrifuge tube, add one-half of the diluted blood volume in lymphatic separation fluid (Lymphoprep, StemCell Technologies);

[0049] 3) Slowly superimpose the diluted blood on the layered liquid surface along the wall of the centrifuge tube, keeping the liquid surface clear;

[0050] 4) Gently put the bone marrow sample mixture centrifuge tube into the centrifuge, and centrifuge at 800g / min for 20min;

[0051] 5) Gently take out the centrifuge tube, layer the bone marrow sample mixture, use a Pasteur pipette to carefully absorb the cells in the middle cloud layer and place them in a new 15mL centrifuge tube;

[0052] 6) Wash twice with serum-free αMEM medium or PBS, and resuspend the isolated cells in medium as spare sample cells;

Embodiment 3

[0053] Example 3: Co-cultivation of primary B-ALL

[0054] B-ALL medium: IMDM medium (HyClone, Thermo Scientific, MA, USA), 10% fetal bovine serum (Gibco, Life Technologies, NY, USA), 2mM L-glutamine 100U / ml penicillin, and 100ug / ml streptomycin ;

[0055] 1) After diluting the spare sample cells and adding appropriate B-ALL medium for dilution, perform cell counting;

[0056] 2) Dilute the backup sample cells to 1×10 according to the cell counting results 6 / mL;

[0057] 3) Aspirate the OP9 cell culture medium planted in the 24-well plate, and wash it once with B-ALL medium;

[0058] 4) Put 1×10 in 2) 6 / mL sample cells were added to the 24-well plate of 3), and 500ul was added to each well;

[0059] 5) After seeding the plate, shake the cells gently to make them evenly spread on the OP9 stromal cells and fully contact with them;

[0060] 6) Placed at 37°C, humidity 95%, 5% CO 2 cultured in an incubator.

[0061] Note: co-cultivation consumes the medium very quickly, ...

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Abstract

The invention belongs to the field of biotechnology, and discloses an in-vitro cell culture method for primary B cell acute lymphoblastic leukemia. According to the in-vitro cell culture method, primary B-ALL cells and OP9 cells are co-cultured so as to maintain the survival rate and proliferation of primary B-ALL cells and greatly relieve the differentiation and senescence of the B-ALL cells. The primary B-ALL cells cultured by adopting the culture method can be proliferated, the cell status is good, and the heterogeneity of tumor cells can be fully embodied. The method is simple, convenient, and economic to operate, and has good application prospects.

Description

technical field [0001] The invention relates to the biological field, in particular to an in vitro culture method for primary B-cell acute lymphoblastic leukemia cells. Background technique [0002] B-cell acute lymphoblastic leukemia (B-ALL, B-cell acute lymphoblastic leukemia), also known as precursor B-cell (pre B-cell) acute lymphoblastic leukemia, is a malignant tumor derived from B-cell progenitor cells. B-ALL is mainly a high-incidence cancer in children, and its incidence rate decreases in adults. Prognosis is good in juvenile patients with B-ALL, and the long-term survival rate (EFS, event-free survival) can reach 90%, but it is poor prognosis and low survival rate in B-ALL adults. In addition, in adult B-ALL patients, the effect of traditional chemotherapy is poor, and the mortality rate is about 60%. Therefore, there is a need to develop new effective treatments for B-ALL. [0003] So far, B-ALL-related research has mainly relied on B-ALL cell lines, and the ce...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/09C12N5/0735C12N5/0775
Inventor 李鹏蒋治武林思妙魏新茹
Owner GUANGZHOU INST OF BIOMEDICINE & HEALTH CHINESE ACAD OF SCI
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