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Method of carrying out isolated culture on immune cells by virtue of peripheral blood

An immune cell, isolation and culture technology, applied in the direction of blood/immune system cells, animal cells, vertebrate cells, etc., to avoid risks and optimize plasma separation conditions

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

[0003] At present, immune cells are mainly cultivated through serum-free medium, but the clinically prescribed serum-free medium needs to add a certain concentration of autologous plasma to achieve a good effect of expansion and culture

Method used

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  • Method of carrying out isolated culture on immune cells by virtue of peripheral blood
  • Method of carrying out isolated culture on immune cells by virtue of peripheral blood
  • Method of carrying out isolated culture on immune cells by virtue of peripheral blood

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] 1. Centrifuge 20ml of peripheral blood at 2000rpm and 4°C for 10min, collect 22ml of autologous plasma, and prepare serum-free medium containing 5% autologous plasma.

[0048] 2. Dilute the lower layer of blood cells with normal saline 1:1, add Ficoll separation solution according to the ratio of 2:1, 700 g Centrifuge for 39min.

[0049] 3. Draw the second layer (from top to bottom) buffy coat layer into another clean centrifuge tube, wash twice with normal saline, which is PBMC.

[0050] 4. Add a certain amount of serum-free medium to make the cell density 1×10 6 / ml, resuspend the cell pellet, inoculate the serum-free medium in the T75 cell culture flask, and add 20U / mL IL-2 and 5ng / mL IL-15.

[0051] 5. After that, take 20ul cell suspension every 3 days for counting, when the cell density is greater than 2×10 6 / ml, add serum-free medium to make the cell density 1×10 6 / ml, the full amount was supplemented with 20U / mL IL-2 and 5ng / mL IL-15.

[0052] 6. On the 14...

Embodiment 2

[0062] 1. Centrifuge 20ml of peripheral blood at 2000rpm and 4°C for 10min, collect 22ml of autologous plasma, and prepare serum-free medium containing 5% autologous plasma.

[0063] 2. Dilute the blood cells in the lower layer with normal saline 1:2, add Ficoll separation solution according to the ratio of 1:2, and centrifuge at 700g for 40min.

[0064] 3. Draw the second layer (from top to bottom) buffy coat layer into another clean centrifuge tube, wash twice with normal saline, which is PBMC.

[0065] 4. Add a certain amount of serum-free medium to make the cell density 5×10 5 / ml, resuspend the cell pellet, inoculate in serum-free medium in a T75 cell culture flask, and add 300U / mL IL-2 and 50ng / mL IL-15.

[0066] 5. After that, take 20ul cell suspension every 3 days for counting, when the cell density is greater than 2×10 6 / ml, add serum-free medium to make the cell density 5×105 / ml, add 300U / mL IL-2 and 50ng / mL IL-15 in full amount.

[0067] 6. On the 14th day of cu...

Embodiment 3

[0069] 1. Centrifuge 20ml of peripheral blood at 2000rpm and 4°C for 10min, collect 22ml of autologous plasma, and prepare serum-free medium containing 5% autologous plasma.

[0070] 2. Dilute the blood cells in the lower layer with normal saline 2:1, add Ficoll separation solution at a ratio of 1:1, and centrifuge at 700g for 20min.

[0071] 3. Draw the second layer (from top to bottom) buffy coat layer into another clean centrifuge tube, wash twice with normal saline, which is PBMC.

[0072] 4. Add a certain amount of serum-free medium to make the cell density 20×10 5 / ml, resuspend the cell pellet, inoculate in serum-free medium in a T75 cell culture flask, and add 100U / mL IL-2 and 30ng / mL IL-15.

[0073] 5. After that, take 20ul cell suspension every 3 days for counting, when the cell density is greater than 2×10 6 / ml, add serum-free medium to make the cell density 20×10 5 / ml, the full amount was supplemented with 100U / mL IL-2 and 30ng / mL IL-15.

[0074] 6. On the 14...

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Abstract

The invention discloses a method of carrying out isolated culture on immune cells by virtue of peripheral blood. The method comprises the following steps: centrifuging the peripheral blood for 10 min under a condition of 2000rpm and 4 DEG C, and collecting the upper-layer plasma to prepare a serum-free medium containing the plasma; adding Ficoll separation liquid and centrifuging; sucking a buffy coat, and washing to obtain PBMC; adding the serum-free medium in the PBMC, inoculating in the serum-free medium, and adding 20-300U / mL of IL-2 and 5-50ng / mL of IL-15; collecting the immune cells while culturing to the 13th to the 15th day. According to the method disclosed by the invention, the centrifuging time, the centrifuging rotational speed and the centrifuging temperature are adjusted, lots of the plasma can be obtained, and the stability of protein content in the plasma and no deficiencies of active factor ingredients can be ensured.

Description

technical field [0001] The invention relates to a method for culturing immune cells, in particular to a method for using peripheral blood to separate and cultivate immune cells. Background technique [0002] 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 tolerance, activate and enhance the body's immune ability, and take into account the dual effects of treatment and health care. Currently, the main cellular immunotherapy therapies include cytokine-induced killer cell (CIK) therapy, dendritic cell (DC) therapy, DC-CIK cell therapy, natural killer cell (NK) therapy, DC-T cell therapy, etc. [0003] At present, immune cells are mainly cultivated through serum-free medium, but the clinically prescribed serum-fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/078
Inventor 陈海佳王一飞葛啸虎应杰罗二梅王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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