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PBMC separation method

A separation liquid, RPMI1640 technology, applied in the field of separation of PBMC, can solve the problems of inconspicuous stratification, large amount of Ficoll separation liquid, and inability to recover plasma, etc., and achieve the effect of shortening separation time, improving yield and purity, and saving time and cost

Inactive Publication Date: 2016-01-20
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, the Ficoll separation method has the disadvantages that plasma cannot be recovered, the amount of Ficoll separation liquid is large, and the layering is not obvious, which needs further improvement.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0024] Transfer 20mL of anticoagulated peripheral blood from healthy people to a 50mL centrifuge tube, centrifuge at 1000g for 10min at 18°C; the peripheral blood in the centrifuge tube is divided into two layers, the upper layer is the first plasma layer, and the lower layer is the blood cell layer. The first plasma layer was centrifuged at 18°C ​​and 2500g for 10min, and the supernatant was filtered through a 0.22μm filter to remove red blood cells, and the filtrate was plasma. Plasma is rich in a large number of protein factors and can be used for subsequent cell culture experiments.

[0025] Dilute the blood cell layer and normal saline at a volume ratio of 1:1.5, and mix thoroughly to form a blood cell dilution. Add the blood cell dilution solution above the Ficoll separation solution in the centrifuge tube, where the volume ratio of the Ficoll separation solution to the blood cell dilution solution is 1:2, and centrifuge for 30 minutes at 18°C, 600g, and 0 centrifugal fo...

Embodiment 2

[0028] Transfer 20mL of anticoagulated peripheral blood from healthy people to a 50mL centrifuge tube, centrifuge at 800g for 10min at 18°C; the peripheral blood in the centrifuge tube is divided into two layers, the upper layer is the first plasma layer, and the lower layer is the blood cell layer. The first plasma layer was centrifuged at 18°C ​​and 3000g for 10 min, and the supernatant was filtered through a 0.22 μm filter to remove red blood cells, and the filtrate was plasma. Plasma is rich in a large number of protein factors and can be used for subsequent cell culture experiments.

[0029] Dilute the blood cell layer and normal saline at a volume ratio of 1:2, and mix thoroughly to form a blood cell dilution. Add the blood cell dilution solution above the Ficoll separation solution in the centrifuge tube, where the volume ratio of the Ficoll separation solution to the blood cell dilution solution is 1:2, and centrifuge for 25 minutes at 18°C, 800g, and 0 centrifugal for...

Embodiment 3

[0032] Transfer 20mL of anticoagulated peripheral blood from healthy people to a 50mL centrifuge tube and centrifuge at 600g for 10min at 18°C; the peripheral blood in the centrifuge tube is divided into two layers, the upper layer is the first plasma layer, and the lower layer is the blood cell layer. The first plasma layer was centrifuged at 18°C ​​and 3000g for 10 min, and the supernatant was filtered through a 0.22 μm filter to remove red blood cells, and the filtrate was plasma. Plasma is rich in a large number of protein factors and can be used for subsequent cell culture experiments.

[0033] Dilute the blood cell layer and normal saline at a volume ratio of 1:2.5, and mix thoroughly to form a blood cell dilution. Add the blood cell dilution solution above the Ficoll separation solution in the centrifuge tube, where the volume ratio of the Ficoll separation solution to the blood cell dilution solution is 1:2, and centrifuge for 20 minutes at 18°C, 1000g, and 0 centrifug...

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PUM

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Abstract

The invention relates to a PBMC separation method. The method includes the following steps that after whole blood centrifugation blood cells are taken for being made into blood cell diluent, a Ficoll separating medium is utilized for conducting centrifugation for 20-30 minutes for separating PBMC in the blood cell diluents, wherein the temperature is 15-18 DEG C, the weight is 500-1000 g, and the centrifugal force ascending and descending speed is zero. According to the PBMC separation method, when the PBMC is separated, the centrifugal force ascending and descending speed is zero so that PBMC layers can be layered more obviously, and it is beneficial to improve the yield and purity of PBMC. By means of the method, plasma can be recycled, and the plasma contains a great number of protein factors which provide rich nutrition for subsequent culture.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for separating PBMCs. Background technique [0002] Cellular immunotherapy is an emerging and new anti-tumor treatment method with significant curative effect in tumor rehabilitation medicine. It makes up for the disadvantages of traditional surgery, radiotherapy, side effects, and chemotherapy. It has been recognized as one of the comprehensive tumor treatment models in the 21st century. It is the most active and promising treatment method, and it is also the only treatment method in the world that has the hope of completely eradicating tumor cells. [0003] Cellular immunotherapy is to collect the body's own immune cells, cultivate them in vitro, increase their number by thousands of times, and enhance their targeted killing function, and then reinfuse them back into the human body to kill pathogens, cancer cells, and mutations in blood and tissues. cells, break immune to...

Claims

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Application Information

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IPC IPC(8): C12N5/0786
Inventor 葛啸虎陈海佳王一飞曾维杰
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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