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Peripheral blood mononuclear cell serum-free freezing medium and freezing method

A cryopreservation method and nuclear cell technology, applied in the field of serum-free cryopreservation and cryopreservation of peripheral blood mononuclear cells, can solve the problems that the cell culture medium cannot be directly injected into the human body, affect the effect of cell therapy, and damage the respiratory system, etc. Achieve the effect of avoiding the risk of spreading potential pathogens, improving safety and reducing damage

Active Publication Date: 2015-06-24
GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] In order to avoid virus infection caused by the use of serum, some people use human serum albumin, but human serum albumin may cause some adverse reactions clinically, such as anaphylactic shock, fever, heart damage, respiratory system damage, kidney damage, etc. Limiting the safety of cell cryopreservation solution containing albumin in clinical practice
[0008] In addition, the existing cryopreservation formula contains cell culture medium, which cannot be directly injected into the human body. Therefore, if the cells need to be transplanted directly after recovery, the cell culture medium must be removed by centrifugation, and centrifugation will cause just The resuscitated cells are damaged and the viability is reduced, which affects the effect of cell therapy

Method used

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  • Peripheral blood mononuclear cell serum-free freezing medium and freezing method
  • Peripheral blood mononuclear cell serum-free freezing medium and freezing method
  • Peripheral blood mononuclear cell serum-free freezing medium and freezing method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0032] A method for isolating human peripheral blood mononuclear cells, comprising the following steps:

[0033] 1. Isolation of PBMCs

[0034] 1.1 Use a 10mL disposable pipette to transfer 30ml of peripheral blood added with sodium citrate anticoagulant to a 50mL centrifuge tube, and centrifuge at 800g for 10min.

[0035] 1.2 After centrifugation, use a Pasteur pipette to suck off the upper layer of plasma, use a 10mL disposable pipette to add 20ml of normal saline to dilute, and mix well.

[0036] 1.3 Take another new 50mL centrifuge tube, add 12mL lymphocyte separation solution (blood dilution solution: lymphatic separation solution = 2:1) into each tube with a 10mL disposable pipette, and use a 10mL disposable pipette to dilute the The blood slowly transfers to the surface of the lymphocyte separation medium, so that a clear interface is formed between the two.

[0037] 1.4 Centrifuge at 700g for 30min, and change the centrifuge lift rate to 0.

[0038] 1.5 After centri...

Embodiment 2

[0043] The serum-free cryopreservation solution for human peripheral blood mononuclear cells is prepared by mixing 5-20% dimethyl sulfoxide, 0.5-10% dextran 40 and the balance Bomali A by volume percentage.

[0044] Component A of Bomaili is: every 1000ml contains 5.26g of sodium chloride, 5.02g of sodium gluconate, 3.68g of sodium acetate, 0.37g of potassium chloride, and 0.30g of magnesium chloride.

[0045] In a further specific scheme, the above-mentioned serum-free cryopreservation solution for human peripheral blood mononuclear cells can be prepared by volume percentage, containing 20% ​​DMSO, 2% dextran 40, and the balance being Bomalil A. The above serum-free cryopreservation solution for human peripheral blood mononuclear cells can also be prepared by volume percentage, containing 10% DMSO, 1% dextran 40, and the balance being Bomali A.

Embodiment 3

[0047] The serum-free cryopreservation method of human peripheral blood mononuclear cells, the specific steps are:

[0048] 1. Take 10-20 ml of the cell cryopreservation solution prepared in Example 2 (20% DMSO+2% dextran 40+Bomalis A).

[0049] 2. Take the PBMC cell pellet obtained by separating in Example 1.

[0050] 4. Add Bomalis A to the PBMC cell pellet to make the cell density 1~2×10 7 / ml, mix by gently pipetting.

[0051] 5. Slowly add the cell freezing solution prepared in step 1 equal to the volume of the cell suspension in the tube to the above PBMC cell suspension along the tube wall, and gently blow and mix to make the cell density (0.5~1)×10 7 / ml.

[0052] 6. Divide the cell suspension into cryopreservation tubes, 1ml per tube.

[0053] 7. Make a mark on the cryopreservation tube, including the umbilical cord number, cell generation number, cell cryopreservation batch number, date of cryopreservation, operator and other relevant information.

[0054] 8. Pl...

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Abstract

The invention discloses a peripheral blood mononuclear cell serum-free freezing medium. The peripheral blood mononuclear cell serum-free freezing medium is prepared from the following components in percentage by volume: 5 to 20% of dimethyl sulfoxide, 0.5 to 10% of dextran 40, and the balance of plasmalyte A. The invention further discloses a method for freezing peripheral blood mononuclear cells through the peripheral blood mononuclear cell serum-free freezing medium. Compared with the prior art, the freezing medium is free of animal serum, human serum and a cell culture medium, and has a good freezing effect; the cells can be directly applied to the clinic after resuscitation and can be also induced in vitro into immune cells NKT and CIK and have high application value in the clinic.

Description

technical field [0001] The invention relates to the technical field of cell cryopreservation liquid, in particular to a serum-free cryopreservation liquid and a cryopreservation method for peripheral blood mononuclear cells. Background technique [0002] Peripheral blood mononuclear cell (PBMC) refers to cells with a single nucleus in peripheral blood, including lymphocytes, monocytes, dendritic cells, and hematopoietic stem cells. PBMC can be induced to differentiate into a variety of immune cells in vitro, such as cytokine-induced killer cells (CIK), natural killer cells (NK), and natural killer T cells (NKT), which have important uses in anti-tumor, anti-infection, etc. . Therefore, cryopreservation of PBMC is of great significance in cell therapy. [0003] Cell cryopreservation is one of the main methods for long-term preservation of cells. Using cryopreservation technology to store cells in -196°C liquid nitrogen at low temperature can temporarily remove the cells fr...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 葛啸虎陈海佳王一飞戴国胜王小燕罗二梅
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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