Method for separating and culturing peripheral blood mononuclear cells

A culture method and cell culture technology, which is applied in the field of isolation and culture of peripheral blood mononuclear cells, can solve the problems that cultured cells are prone to disease, and achieve the effect of pure cells, preventing excessive activation, and ensuring safety

Inactive Publication Date: 2020-10-30
KUNMING MEDICAL UNIVERSITY
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] Aiming at the deficiencies of the prior art, the present invention provides a method for separating and culturing peripheral blood mononuclear cells, which solves the problem that the cultured cells are prone to disease

Method used

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Embodiment 1

[0026] An embodiment of the present invention provides a method for isolating and culturing peripheral blood mononuclear cells, comprising the following steps:

[0027] S1. Collect peripheral blood: extract peripheral blood in a sterile environment, and add antihemagglutinin to it;

[0028] S2. Centrifugal separation: Dilute the collected peripheral blood, then add polysucrose-diatrizoate layering liquid to it, and separate peripheral blood mononuclear cells by polysucrose-diatrizoate density gradient centrifugation, Make the blood form obvious stratification;

[0029] S3. Extracting peripheral blood mononuclear cells: use a capillary pipette to absorb the gray-white layer in the layer, suck the mononuclear cells into the capillary pipette, and extract peripheral blood mononuclear cells;

[0030] S4. Purifying peripheral blood mononuclear cells: washing by adding balanced salt solution, and removing mixed platelets by secondary centrifugation to obtain purified peripheral blo...

Embodiment 2

[0037] An embodiment of the present invention provides a method for isolating and culturing peripheral blood mononuclear cells, comprising the following steps:

[0038] S1. Collect peripheral blood: extract peripheral blood in a sterile environment, and add antihemagglutinin to it;

[0039] S2. Centrifugal separation: Dilute the collected peripheral blood, then add polysucrose-diatrizoate layering liquid to it, and separate peripheral blood mononuclear cells by polysucrose-diatrizoate density gradient centrifugation, Make the blood form obvious stratification;

[0040] S3. Extracting peripheral blood mononuclear cells: use a capillary pipette to absorb the gray-white layer in the layer, suck the mononuclear cells into the capillary pipette, and extract peripheral blood mononuclear cells;

[0041] S4. Purifying peripheral blood mononuclear cells: washing by adding balanced salt solution, and removing mixed platelets by secondary centrifugation to obtain purified peripheral blo...

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Abstract

The invention provides a method for separating and culturing peripheral blood mononuclear cells, and relates to the field of biotechnology cell culture. The separation and culture method of the peripheral blood mononuclear cells comprises the following steps: S1, collecting peripheral blood, S2, carrying out centrifugal separation, S3, extracting peripheral blood mononuclear cells, S4, purifying the peripheral blood mononuclear cells, S5, preparing a cell culture medium, S6, carrying out cell culture, and S7, performing preservation. An ALK inhibitor and a GSK-3 inhibitor are arranged, cytopathy in a cell culture process can be inhibited, excessive activation of pathological cells is prevented and rapid proliferation is carried out, animal serum is not added in the culture process, so thatthe possibility of obtaining viruses from animals in the culture process is reduced, the safety in the culture process is ensured, and peripheral blood mononuclear cells are cleaned and purified formultiple times after separation is completed, so that the cultured cells basically do not contain other impurity cells, and the cultured cells are relatively pure.

Description

technical field [0001] The invention relates to the field of biotechnology cell culture, in particular to a method for separating and culturing peripheral blood mononuclear cells. Background technique [0002] Peripheral blood is blood other than bone marrow, which is rich in sources and convenient to collect. Immune cells isolated and cultured from peripheral blood have the characteristics of fast proliferation, safety, persistence, adaptability, and systemicity. Compared with traditional radiotherapy and other methods, active immunization can stimulate systemic anti-tumor effects. More extensive, especially suitable for malignant tumors with multiple lesions or extensive metastases; side effects are minimal. Peripheral blood mononuclear cells are cells in the peripheral blood that have a single nucleus, including lymphocytes and monocytes. [0003] During the culture process of peripheral blood mononuclear cells, due to some changes in light, temperature and environment,...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2500/12C12N2500/25C12N2500/32C12N2500/38C12N2501/10C12N2501/727C12N2501/998C12N2509/00
Inventor 黄宁郑永唐郑昌博杨柳萌罗荣华韦海容王婷李俊洁
Owner KUNMING MEDICAL UNIVERSITY
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