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Separation method of human peripheral blood mononuclear cells

A separation method, the technology of human peripheral blood, applied in the field of cell separation, can solve the problems of high operation, influence on cell quantity and purity, etc., and achieve the effect of good differentiation ability, high expansion multiple in vitro, high cell viability and purity

Inactive Publication Date: 2015-04-01
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The purity of the cells obtained by this method is relatively high, but the requirements for operation are relatively high. If the operation is not careful enough, the number and purity of the isolated cells will be greatly affected.

Method used

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  • Separation method of human peripheral blood mononuclear cells
  • Separation method of human peripheral blood mononuclear cells
  • Separation method of human peripheral blood mononuclear cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0034] 1. Wipe and disinfect the work surface of the biological safety cabinet with 75% ethanol, and put the anticoagulant tube containing peripheral blood into the ultra-clean bench.

[0035] 2. Transfer the peripheral blood in the anticoagulant tube to a centrifuge tube, and centrifuge at 2000rpm for 10 minutes. After centrifugation, it is divided into two layers, the upper layer is plasma, and the lower layer is blood cell sediment. The upper layer of plasma was collected in another centrifuge tube, marked and placed in a 4°C refrigerator for later use.

[0036] 3. Transfer the blood cells in the lower layer to a 50 mL centrifuge tube, add an equal volume of 6% g / mL hydroxyethyl starch solution, mix well and let it settle naturally for 20 minutes.

[0037] 4. Take another new 50mL centrifuge tube, and add Ficoll lymphocyte separation medium (brand: Axis-shield; supplier: Shenzhen Dakowei Biotechnology Co., Ltd.; article number: AS1114546) to each tube, and after natural sed...

Embodiment 2

[0044] 1. Wipe and disinfect the work surface of the biological safety cabinet with 75% ethanol, and put the anticoagulant tube containing peripheral blood into the ultra-clean bench.

[0045] 2. Transfer the peripheral blood in the anticoagulant tube to a centrifuge tube, and centrifuge at 1500rpm for 8 minutes. After centrifugation, it is divided into two layers, the upper layer is plasma, and the lower layer is blood cell sediment. The upper layer of plasma was collected in another centrifuge tube, marked and placed in a 4°C refrigerator for later use.

[0046] 3. Transfer the blood cells in the lower layer to a 50mL centrifuge tube, add an equal volume of 4% g / mL hydroxyethyl starch solution, mix well and allow it to settle naturally for 15 minutes.

[0047] 4. Take another new 50mL centrifuge tube, and add Ficoll lymphocyte separation medium (brand: Axis-shield; supplier: Shenzhen Dakowei Biotechnology Co., Ltd.; article number: AS1114546) to each tube, and after natural ...

Embodiment 3

[0054] 1. Wipe and disinfect the work surface of the biological safety cabinet with 75% ethanol, and put the anticoagulant tube containing peripheral blood into the ultra-clean bench.

[0055] 2. Transfer the peripheral blood in the anticoagulant tube to a centrifuge tube, and centrifuge at 3000rpm for 12 minutes. After centrifugation, it is divided into two layers, the upper layer is plasma, and the lower layer is blood cell sediment. The upper layer of plasma was collected in another centrifuge tube, marked and placed in a 4°C refrigerator for later use.

[0056] 3. Transfer the blood cells in the lower layer to a 50mL centrifuge tube, add an equal volume of 10% g / mL hydroxyethyl starch solution, mix well and let it settle naturally for 35min.

[0057] 4. Take another new 50mL centrifuge tube, and add Ficoll lymphocyte separation medium (brand: Axis-shield; supplier: Shenzhen Dakowei Biotechnology Co., Ltd.; article number: AS1114546) to each tube, and after natural sediment...

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Abstract

The invention discloses a separation method of human peripheral blood mononuclear cells. The separation method combines the advantages of a hydroxyethyl starch precipitation method and a Ficoll density gradient centrifugation method. The PBMC with a purity of greater than 95% and a vitality of greater than 85% is obtained by removing most of red blood cells by the hydroxyethyl starch precipitation method, then naturally settling in air, and then further purifying the mononuclear cells by the Ficoll density gradient centrifugation method, aiming at the separation characteristic of a high-capacity blood sample. Compared with a method without natural setting in air, the vitality and the purity of the cells obtained by the separation method are higher.

Description

technical field [0001] The invention relates to a method for separating cells, in particular to a method for separating human peripheral blood mononuclear cells. Background technique [0002] Cell therapy is a new technology for disease treatment that has emerged in recent years. It refers to the use of the characteristics of certain cells with specific functions, which are obtained by bioengineering methods and / or processed by in vitro expansion and special culture to enhance the ability of these cells. Immunity, killing pathogens and tumor cells, promoting tissue and organ regeneration and body recovery and other therapeutic effects, so as to achieve the purpose of treating diseases. [0003] With its good curative effect, less side effects, more individualized and individualized unique advantages, cell therapy provides a choice, sometimes even the last choice, for the treatment of some refractory diseases. Cell therapy will play an important role in clinical treatment bo...

Claims

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Application Information

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IPC IPC(8): C12N5/078
Inventor 陈海佳王一飞葛啸虎陈洁鸿王小燕罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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