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Separation method of PBMCs (peripheral blood mononuclear cells)

A separation method and peripheral blood technology, applied in the field of regenerative medicine, can solve the problems of low yield and low activity, and achieve the effect of large quantity and high activity

Pending Publication Date: 2019-12-10
GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Based on this, it is necessary to provide a method for isolating peripheral blood mononuclear cells to solve the problems of low yield and low activity of existing methods for isolating peripheral blood mononuclear cells

Method used

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  • Separation method of PBMCs (peripheral blood mononuclear cells)
  • Separation method of PBMCs (peripheral blood mononuclear cells)

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] A method for isolating peripheral blood mononuclear cells, comprising the following steps:

[0027] 1) Peripheral blood collection: 70mL of human peripheral blood will be extracted and placed in a sodium heparin blood collection tube. After the collection is completed, the sodium heparin blood collection tube will be placed in a sealed sterile transport bottle for refrigerated transportation at a temperature of 4-8°C;

[0028] 2) Pretreatment: After the peripheral blood arrives in the laboratory, the experimenters operate in a sterile environment, put the heparin sodium blood collection tube in the centrifuge for centrifugation, the centrifugation speed is 1800rpm, the time is 10min, the rising speed is 7 and the falling speed is 10min. 1. Separate the upper layer of blood and the lower layer of blood cells; extract the upper layer of plasma for storage, transfer the lower layer of blood cells to a 100mL transfer bag, add hydroxyethyl starch, the volume ratio of the lowe...

Embodiment 2

[0032] A method for isolating peripheral blood mononuclear cells, comprising the following steps:

[0033] 1) Peripheral blood collection: 70mL of human peripheral blood will be extracted and placed in a sodium heparin blood collection tube. After the collection is completed, the sodium heparin blood collection tube will be placed in a sealed sterile transport bottle for refrigerated transportation at a temperature of 4-8°C;

[0034] 2) Pretreatment: After the peripheral blood arrives in the laboratory, the experimenter operates in a sterile environment, puts the heparin sodium blood collection tube in a centrifuge and centrifuges at a speed of 1700 rpm for 12 minutes to separate the upper blood and lower blood cells; Extract the upper layer of plasma for storage, transfer the lower layer of blood cells to a 100mL transfer bag, add hydroxyethyl starch, the volume ratio of the lower layer of blood cells to the hydroxyethyl starch solution is 3:1, mix well, settle for 65 minutes,...

Embodiment 3

[0038] A method for isolating peripheral blood mononuclear cells, comprising the following steps:

[0039] 1) Peripheral blood collection: 70mL of human peripheral blood will be extracted and placed in a sodium heparin blood collection tube. After the collection is completed, the sodium heparin blood collection tube will be placed in a sealed sterile transport bottle for refrigerated transportation at a temperature of 4-8°C;

[0040] 2) Pretreatment: After the peripheral blood arrives in the laboratory, the experimenter operates in a sterile environment, puts the heparin sodium blood collection tube in a centrifuge and centrifuges at a speed of 1900 rpm for 8 minutes to separate the upper blood and lower blood cells; Extract the upper layer of plasma for storage, transfer the lower layer of blood cells to a 100mL transfer bag, add hydroxyethyl starch, the volume ratio of the lower layer of blood cells to the hydroxyethyl starch solution is 3:1, mix well, settle for 65 minutes, an...

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Abstract

The invention provides a separation method of PBMCs (peripheral blood mononuclear cells) and relates to the field of regenerative medicines. The separation method comprises steps as follows: pretreatment: acquired peripheral blood is centrifuged, upper-layer blood and lower-layer blood cells are separated, the lower-layer blood cells and a hydroxyethyl starch solution are mixed uniformly and placed to be settled, a liquid supernatant after settlement is removed, and lower-layer precipitates are reserved; separation: a red blood cell lysis buffer is added to the lower-layer precipitates, uniform mixing and centrifuging are carried out, cell precipitates are taken, PBS is added for re-suspension, the cell suspension is added to a lymphocyte separation liquid, centrifugation is carried out,a liquid supernatant is removed, cell precipitates are reserved, PBS, heparin and human serum albumin are added, mixing and centrifuging are carried out, and PBMC precipitates are obtained; and culture: the PBMC precipitates are resuspended with a culture medium and sub-packed in a culture bottle for culture. With the adoption of the method, the number of the separated PBMCs is large, and the PBMCs are high in purity and activity.

Description

technical field [0001] The invention relates to the field of regenerative medicine, in particular to a separation method and application of peripheral blood mononuclear cells. Background technique [0002] Peripheral Blood Mononuclear Cell (PBMC) is a cell with a single nucleus in peripheral blood, mainly including lymphocytes (T lymphocytes and B lymphocytes), monocytes, phagocytes, dendritic cells and others A small number of cell types that are important in the functioning of the body's immune response. In recent years, the research on peripheral blood mononuclear cells has become more and more in-depth. It has been found that peripheral blood mononuclear cells can proliferate and differentiate in vitro. Compared with extracting immune cells from bone marrow, it is easier to obtain peripheral blood mononuclear cells from peripheral blood, and it is basically the pain caused by the human body. [0003] In 2010, a study showed that PBMCs activated by CD80 combined with CD...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2509/00
Inventor 何美弟江嘉豪王进辉于莉
Owner GUANGDONG VITALIFE BIOTECHNOLOGY CO LTD
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