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Isolated culture method of leukemia variant cells

A technology for separation and culture of leukemia, applied in the field of separation and culture of leukemia mutant cells, can solve the problems of little research and achieve the effect of improving activity, stable genetic traits, and high cell survival rate

Inactive Publication Date: 2021-09-21
上海十指生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, there is little research on the method of isolation and culture of primary leukemia cells, especially leukemia mutant cells

Method used

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  • Isolated culture method of leukemia variant cells
  • Isolated culture method of leukemia variant cells

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Experimental program
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Effect test

preparation example Construction

[0026] (3) Preparation of cell suspension:

[0027] Under sterile conditions, the leukemia variant cells separated in step (2) are added to the complete medium of 10% fetal bovine serum and blown into a single cell suspension;

[0028] (4) Cultivate:

[0029] Inoculate the single-cell suspension in step (3) into a culture bottle, then place it in an incubator for cell culture, and control the culture conditions as a temperature of 25-30°C and a CO of 2-3% saturated humidity. 2 , carry out intermittent ultrasonic-magnetic field coupling treatment at the same time of cultivation, the specific procedure of intermittent ultrasonic-magnetic field coupling treatment is: in the process of cultivating, conduct ultrasonic-magnetic field coupling treatment every 12 to 16 hours, the frequency of ultrasonic wave during treatment The control is 20-30kHz, the strength of the magnetic field is 50-70mT, and the time for each treatment is 30-40min.

Embodiment 1

[0032] A method for isolating and culturing leukemia mutant cells, comprising the steps of:

[0033] (1) Collection of bone marrow fluid:

[0034] Collect 2 mL of bone marrow fluid from untreated leukemia patients through bone marrow aspiration and put it in a heparinized sterile test tube for use;

[0035] (2) Separation:

[0036] Add heparin-anticoagulated bone marrow fluid under sterile conditions to separate lymphocytes, place it on a centrifuge and centrifuge at 2000rpm for 7min, then absorb the middle cell layer and place it in PBS solution for rinsing, then centrifuge at 500rpm for 3min, and finally Centrifuge at 3000rpm for 10 minutes, discard the supernatant after completion, and the remaining precipitate is the isolated leukemia mutant cells;

[0037] (3) Preparation of cell suspension:

[0038] Add the leukemia mutant cells separated in step (2) into the complete culture medium of 10% fetal bovine serum under aseptic conditions and blow to form a single cell susp...

Embodiment 2

[0042] A method for isolating and culturing leukemia mutant cells, comprising the steps of:

[0043] (1) Collection of bone marrow fluid:

[0044] Collect 2.5 mL of bone marrow fluid from untreated leukemia patients through bone marrow aspiration and put it in a heparinized sterile test tube for use;

[0045] (2) Separation:

[0046] Add heparin-anticoagulated bone marrow fluid under sterile conditions to separate lymphocytes, place it on a centrifuge for 8 minutes at 2500 rpm, then absorb the middle cell layer and place it in PBS solution for rinsing, then centrifuge at 550 rpm for 4 minutes, and finally Centrifuge at 3500rpm for 11.5min, discard the supernatant after completion, and the remaining precipitate is the isolated leukemia mutant cells;

[0047] (3) Preparation of cell suspension:

[0048] Add the leukemia mutant cells separated in step (2) into the complete culture medium of 10% fetal bovine serum under aseptic conditions and blow to form a single cell suspensi...

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Abstract

The invention discloses an isolated culture method of leukemia variant cells, and belongs to the technical field of cell culture. The method comprises the following steps of: (1) collecting marrow fluid; (2) separating; (3) preparing a cell suspension; and (4) culturing. The invention provides an isolated culture method of leukemia variant cells. The cells isolated and cultured by the method have high cell survival rate. The cell state is the best in 24 hours. Although part of cells die in 48-72 hours, the difference has no statistical significance. The isolated cells are similar to in-vivo cells in biological characters and stable in genetic character. A good cell source is provided for leukemia biology, cytogenetics, in-vitro induced differentiation and the like.

Description

technical field [0001] The invention belongs to the technical field of cell culture, and in particular relates to a method for separating and culturing leukemia mutant cells. Background technique [0002] Leukemia patients relapse, have a high mortality rate, and cannot survive for a long time, because the current clinical chemotherapy drugs are all aimed at the leukemia cells in the proliferating stage. Although the leukemia condition can be regressed or remitted, the remaining leukemia stem cells cannot be effectively removed, which leads to The root cause of leukemia recurrence cannot be eliminated, and because the toxic side effects of chemotherapy itself reduce the autoimmunity of leukemia patients, laying hidden dangers for leukemia recurrence. [0003] For the research on leukemia, in the past, cell lines were mostly used as the research object, but cell lines are prone to variation after multiple passages, while primary cultured cells are similar in biological shape ...

Claims

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Application Information

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IPC IPC(8): C12N5/09C12N13/00
CPCC12N5/0694C12N13/00C12N2509/00C12N2521/10C12N2529/00
Inventor 陈彦光
Owner 上海十指生物科技有限公司
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