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Method for extracting immune cells from marrow

A technology of immune cells and bone marrow cells, applied in the field of cell biology, can solve problems such as unfavorable clinical application and cytotoxicity, and achieve high safety, small cell damage, and good cell protection effect

Inactive Publication Date: 2017-08-04
DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

In the prior art, conventional Ficoll 400 separation fluid has certain toxicity to cells, which is not conducive to clinical application

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0065] A method for extracting immune cells from bone marrow, comprising the steps of:

[0066] (1) Collect human bone marrow, dilute it 2 times with α-MEM culture medium containing 10% fetal bovine serum, centrifuge at 200rpm for 4min, discard the supernatant and fat layer to obtain bone marrow cells;

[0067] (2) Transfer human peripheral blood into a centrifuge tube, centrifuge at 500rpm for 9 minutes, absorb the upper layer of plasma for use, and the lower layer of cells is used to separate peripheral blood lymphocytes; α-MEM culture medium was made into cell suspension;

[0068] (3) Add the separation solution into the centrifuge tube; spread the cell suspension on the separation solution, centrifuge at 600rpm for 20min, absorb buffy coat cells to obtain immune cells;

[0069] (4) Add an equal volume of immune cells to the immune cell cryopreservation solution and mix evenly to obtain a cell resuspension, divide the cell resuspension into cryopreservation bags, and then ...

Embodiment 2

[0085] A method for extracting immune cells from bone marrow, comprising the steps of:

[0086] (1) Collect human bone marrow, dilute it 2.5 times with α-MEM culture medium containing 11% fetal bovine serum, centrifuge at 250 rpm for 3.5 min, discard the supernatant and fat layer, and obtain bone marrow cells;

[0087] (2) Transfer human peripheral blood into a centrifuge tube, centrifuge at 550rpm for 8 minutes, absorb the upper layer of plasma for use, and the lower layer of cells is used to separate peripheral blood lymphocytes; α-MEM culture medium was made into cell suspension;

[0088] (3) Add the separation solution into the centrifuge tube; spread the cell suspension on the separation solution, centrifuge at 650rpm for 18min, absorb buffy coat cells to obtain immune cells;

[0089] (4) Add an equal volume of immune cells to the immune cell cryopreservation solution and mix evenly to obtain a cell resuspension, divide the cell resuspension into cryopreservation bags, a...

Embodiment 3

[0105] A method for extracting immune cells from bone marrow, comprising the steps of:

[0106] (1) Collect human bone marrow, dilute it 3 times with α-MEM culture medium containing 12% fetal bovine serum, centrifuge at 300rpm for 3min, discard the supernatant and fat layer to obtain bone marrow cells;

[0107] (2) Transfer human peripheral blood into a centrifuge tube, centrifuge at 600rpm for 7 minutes, absorb the upper layer of plasma for later use, and the lower layer of cells is used to separate peripheral blood lymphocytes; α-MEM culture medium was made into cell suspension;

[0108] (3) Add the separation solution into the centrifuge tube; spread the cell suspension on the separation solution, centrifuge at 700rpm for 15min, absorb buffy coat cells to obtain immune cells;

[0109] (4) Add an equal volume of immune cells to the immune cell cryopreservation solution and mix evenly to obtain a cell resuspension, divide the cell resuspension into cryopreservation bags, and...

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Abstract

The invention specifically relates to a method for extracting immune cells from marrow, belonging to the technical field of cytobiology. The method comprises the following steps: (1) acquiring human marrow, diluting the human marrow with an alpha-MEM nutrient solution containing fetal calf serum, then carrying out centrifugation and discarding supernatant and a fat layer so as to obtain marrow cells; (2) transferring human peripheral blood into a centrifuge tube, carrying out centrifugation, sucking up blood plasma at an upper layer for subsequent usage and cells at a lower layer for separation of peripheral blood lymphocytes, and adding an alpha-MEM nutrient solution containing the blood plasma at the upper layer into the marrow cells to prepare a cell suspension; (3) adding a separating medium into another centrifuge tube, flatly spreading the cell suspension onto the separating medium, carrying out centrifugation, and sucking up albuginea-layer cells so as to obtain immune cells; and (4) adding isopyknic immune cells into an immune cell cryopreservation solution, carrying out uniform mixing and carrying out immune cell cryopreservation. The method provided by the invention can effectively improve the separation rate of the immune cells and the survival rate of the immune cells after cell cryopreservation and enhances the security of cells transfused back to a patient.

Description

technical field [0001] The invention relates to the technical field of cell biology, in particular to a method for extracting immune cells from bone marrow. Background technique [0002] Bone marrow is an important immune organ of the human body and contains a large number of immune cells. During clinical application, a common problem is that it is difficult to ensure a sufficient number of immune cells. At present, the cryopreservation technology is mainly used to preserve immune cells, and when needed, they can be revived and induced to culture into various immune cells. However, during the process of separation and cryopreservation, individual cells will be damaged or even toxic, especially during the cryopreservation process, the thermodynamic, chemical and physical environment of cells will be significantly changed, accompanied by the risk of biological damage. [0003] On the other hand, the bone marrow contains a lot of fat, which affects the separation effect and ca...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078A01N1/02
CPCA01N1/0221C12N5/0634C12N2509/00
Inventor 罗天恩
Owner DONGGUAN BOALAI BIOLOGICAL TECH CO LTD
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