Method for separating lymphocytes from leukocyte removal filter

A lymphocyte and leukocyte depletion technology, applied in animal cells, vertebrate cells, blood/immune system cells, etc., can solve the problem of lymphocyte waste and other problems, and achieve the effect of avoiding waste.

Inactive Publication Date: 2014-08-13
SHENZHEN KIVITA INNOVATIVE DRUG INST +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

At present, most of the methods used at home and abroad are to use the leukocyte-removing filter to remove the leukocytes in the blood. This method can make 99% of the leukocytes remain in the leukocyte-removing filter, of which lymphocytes account for a large proportion, which will cause lymphocytes. waste of cells

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] This example provides a method for isolating lymphocytes from a leukocyte-removing filter:

[0024] 1. Isolation of lymphocytes

[0025] 1) Obtain a 2-unit (400ml) leukocyte-removing filter from the Shenzhen Blood Center. After the blood bank uses the leukocyte-removing filter to filter 400ml of whole blood (normal blood donation), 99% of the leukocytes remain in the filter.

[0026] 2) Rinse the leukocyte-removing filter with 60 ml of 1×PBS to obtain a mixture of PBS, leukocytes and part of the blood.

[0027] 3) Centrifuge the mixture of PBS, white blood cells and part of the blood at 1500 rpm for 20 minutes to discard the plasma.

[0028] 4) Add 30 ml of 1×PBS to the cell mixture from which the plasma was discarded, and mix carefully to form a mixture.

[0029] 5) Carefully superimpose the mixed solution on the liquid surface of the cell separation solution (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LTS1077), mixed solu...

Embodiment 2

[0036] This example provides a method for isolating lymphocytes from a leukocyte-removing filter:

[0037] 1. Isolation of lymphocytes

[0038] 1) Obtain a 2-unit (400ml) leukocyte-removing filter from the Shenzhen Blood Center. After the blood bank uses the leukocyte-removing filter to filter 400ml of whole blood (normal blood donation), 99% of the leukocytes remain in the filter.

[0039] 2) Rinse the leukocyte-removing filter with 50 ml of 1×PBS to obtain a mixture of PBS, leukocytes and part of the blood.

[0040] 3) Centrifuge the mixture of PBS, white blood cells and part of the blood at 2000 rpm for 15 minutes to discard the plasma.

[0041] 4) Add 30 ml of 1×PBS to the cell mixture from which the plasma was discarded, and mix carefully to form a mixture.

[0042] 5) Carefully superimpose the mixed solution on the liquid surface of the cell separation solution (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LTS1077), the mixed ...

Embodiment 3

[0049] This example provides a method for isolating lymphocytes from a leukocyte-removing filter:

[0050] 1. Isolation of lymphocytes

[0051] 1) Obtain a 2-unit (400ml) leukocyte-removing filter from the Shenzhen Blood Center. After the blood bank uses the leukocyte-removing filter to filter 400ml of whole blood (normal blood donation), 99% of the leukocytes remain in the filter.

[0052] 2) Rinse the leukocyte-removing filter with 80 ml of 1×PBS to obtain a mixture of PBS, leukocytes and part of the blood.

[0053] 3) Centrifuge the mixture of PBS, white blood cells and part of the blood at 2000 rpm for 20 minutes to discard the plasma.

[0054] 4) Add 40 ml of 1×PBS to the cell mixture from which the plasma has been discarded, and mix carefully to form a mixture.

[0055] 5) Carefully superimpose the mixed solution on the liquid surface of the cell separation solution (purchased from Tianjin Haoyang Biological Products Technology Co., Ltd., product number LTS1077), mixed...

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PUM

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Abstract

The invention provides a method for separating lymphocytes from a leukocyte removal filter. The method comprises the following steps: obtaining mixed liquid of PBS (Phosphate Buffered Saline), leukocytes and a part of blood; discarding plasma, adding the phosphate buffered saline to the cell mixed liquid without the plasma, and carefully and uniformly mixing so as to form mixed liquid; and carefully superimposing the mixed liquid on cell separating liquid, centrifuging, collecting a second layer of cells, putting in the phosphate buffered saline, fully and uniformly mixing, centrifuging, discharging supernatant, remaining precipitates, then repeatedly washing, and finally precipitating so as to obtain separated peripheral blood mononuclear cells; and culturing the separated peripheral blood mononuclear cells in a 37-DEG C incubator with 5% of CO2, cultivating overnight, and collecting the suspended lymphocytes. The method for separating the lymphocytes from the leukocyte removal filter, which is provided by the invention, is capable of avoiding the wasting of the lymphocytes and has no influence on other components such as red blood cells and blood platelets; the number of the lymphocytes obtained by using the separation method can reach 1*10<6>, and the survival rate of the lymphocytes is greater than 90%.

Description

technical field [0001] The invention relates to a method for separating lymphocytes, in particular to a method for separating lymphocytes from a leukocyte-removing filter. Background technique [0002] T lymphocytes are derived from pluripotent stem cells in the bone marrow. During the embryonic and primary stages of the human body, some pluripotent stem cells or pre-T cells in the bone marrow migrate to the thymus, differentiate and mature under the induction of thymus hormones, and become immune-competent T cells. cell. Mature T cells are distributed to the thymus-dependent area of ​​peripheral immune organs to settle in the thymus-dependent area through the blood flow, and recirculate through lymphatic vessels, peripheral blood, and tissue fluid to exert cellular immunity and immune regulation functions. Cytotoxic T cells function like a "killer" or cytotoxin in that they kill target cells that respond to specific antigens. The main surface marker of cytotoxic T cells i...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
Inventor 蒋宇扬陈妍
Owner SHENZHEN KIVITA INNOVATIVE DRUG INST
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