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Method for extracting immune cells from peripheral blood

A technology of immune cells and peripheral blood, applied in the biological field, can solve problems such as unfavorable clinical application and cytotoxicity, and achieve the effects of reducing immune rejection, high safety, and small uncertainty of raw materials

Inactive Publication Date: 2019-09-13
北京弘润天源基因生物技术有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] In the prior art, conventional Ficoll 400 separation fluid has certain toxicity to cells, which is not conducive to clinical application, and the survival rate of cells after cryopreservation and recovery is usually between 70% and 90%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0023] 1) Pretreatment of peripheral blood: transfer the peripheral blood added with anticoagulant into a centrifuge tube, centrifuge at 750g for 10 minutes, absorb the upper layer of plasma for later use, add an equal volume of PBS solution to the lower layer of cells, and mix well to obtain a cell suspension;

[0024] 2) adding the separation liquid containing trehalose, ultra-low viscosity sodium alginate and sodium diatrizoate into the centrifuge tube;

[0025] 3) spreading the cell suspension on the centrifugate, centrifuging at 8-25°C, 500g-800g, 10-30min, absorbing buffy coat cells to obtain immune cells;

[0026] 4) Add an equal volume of the immune cells to the cryopreservation solution and mix them evenly, and distribute them into cell cryopreservation tubes, place the cell cryopreservation tubes in a programmed cryopreservation box, freeze at -80°C first, and then Transfer to liquid nitrogen and freeze.

[0027] Wherein, the formula of the cryopreservation solution...

Embodiment 2

[0029] 1) Pretreatment of peripheral blood: transfer the peripheral blood added with anticoagulant into a centrifuge tube, centrifuge at 750g for 10 minutes, absorb the upper layer of plasma for later use, add an equal volume of PBS solution to the lower layer of cells, and mix well to obtain a cell suspension;

[0030] 2) adding the separation liquid containing trehalose, ultra-low viscosity sodium alginate and sodium diatrizoate into the centrifuge tube;

[0031] 3) spreading the cell suspension on the centrifugate, centrifuging at 8-25°C, 500g-800g, 10-30min, absorbing buffy coat cells to obtain immune cells;

[0032] 4) Add an equal volume of the immune cells to the cryopreservation solution and mix them evenly, and distribute them into cell cryopreservation tubes, place the cell cryopreservation tubes in a programmed cryopreservation box, freeze at -80°C first, and then Transfer to liquid nitrogen and freeze.

[0033] Wherein, the formula of the cryopreservation solution...

Embodiment 3

[0035] 1) Pretreatment of peripheral blood: transfer the peripheral blood added with anticoagulant into a centrifuge tube, centrifuge at 750g for 10 minutes, absorb the upper layer of plasma for later use, add an equal volume of PBS solution to the lower layer of cells, and mix well to obtain a cell suspension;

[0036] 2) adding the separation liquid containing trehalose, ultra-low viscosity sodium alginate and sodium diatrizoate into the centrifuge tube;

[0037] 3) spreading the cell suspension on the centrifugate, centrifuging at 8-25°C, 500g-800g, 10-30min, absorbing buffy coat cells to obtain immune cells;

[0038] 4) Add an equal volume of the immune cells to the cryopreservation solution and mix them evenly, and distribute them into cell cryopreservation tubes, place the cell cryopreservation tubes in a programmed cryopreservation box, freeze at -80°C first, and then Transfer to liquid nitrogen and freeze.

[0039]Wherein, the formula of the cryopreservation solution ...

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Abstract

The invention discloses a method for extracting immune cells from peripheral blood and belongs to the field of biotechnologies. The method includes the following steps that peripheral blood is pretreated, wherein 750 g of the peripheral blood to which anticoagulant is added is transferred into a centrifugal tube, centrifugation is conducted for 10 minutes, upper-layer plasma is absorbed for standby use, a PBS solution of the same size is added to a lower layer of cells, the mixture is mixed to be uniform, and cell suspension is obtained; separation liquid containing anticoagulant, super-low-viscosity sodium alginate and sodium amidotrizoate is added into the centrifugation tube; the cell suspension is flatly laid on centrifugal liquid, 500-800 g of the mixture is subjected to centrifugation at the temperature of 8-25 DEG C for 10-30 minutes, tunica albuginea layer cells are absorbed, and immune cells are obtained; the immune cells of the same size are added to a frozen stock solution to be mixed uniformly, the mixture is put into cell frozen stock tubes in a split mode, and the cell frozen stock tubes are placed into a program frozen stock box, frozen at the temperature of -80 DEG C in the first place and then transferred to liquid nitrogen for frozen stock. All raw materials in the formula meet the standard of an intravenous injection level crude drug.

Description

technical field [0001] The invention relates to the field of biotechnology, in particular to a method for extracting immune cells from peripheral blood. Background technique [0002] Extracting immune cells from human peripheral blood not only has good prospects in the treatment of cancer, but also from the perspective of health preservation, immune cells can also be extracted from peripheral blood of patients when they are healthy, and can be used in the future. When the patient has low immunity or other symptoms, the stored immune cells can be reinfused back to the patient to improve the patient's immunity. At present, single cells in peripheral blood are mainly preserved by cryopreservation technology, and when needed, they are recovered and induced to culture into various immune cells. However, during the process of separation and cryopreservation, individual cells will be damaged or even toxic, especially during the cryopreservation process, the thermodynamic, chemical...

Claims

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Application Information

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Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/078A01N1/02
Inventor 宫喜魁李若鲲马艳马艳玲郝丽敏
Owner 北京弘润天源基因生物技术有限公司
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