Cell cryo-preserved liquid, application, and immune cell cryo-preservation method

A technology of immune cells and cryopreservation methods, which can be used in applications, preservation of human or animal bodies, animal husbandry, etc., and can solve problems such as danger, immune rejection, and infectious diseases.

Active Publication Date: 2015-12-09
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] Most of the existing cell cryopreservation solutions use DMSO combined with animal serum, most of which are 10% DMSO and 90% fetal bovine serum. However, due to the high content of DMSO, it has considerable toxicity and is not good for patients. Bovine serum contains a large amount of foreign proteins, which is not only dangerous for infectious diseases, but also easily causes allergic reactions or immune rejection, so it cannot be directly used for clinical reinfusion

Method used

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  • Cell cryo-preserved liquid, application, and immune cell cryo-preservation method
  • Cell cryo-preserved liquid, application, and immune cell cryo-preservation method
  • Cell cryo-preserved liquid, application, and immune cell cryo-preservation method

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Experimental program
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Effect test

Embodiment 1

[0056] 1.1 Take 20ml of peripheral blood or umbilical cord blood, add an equal volume of normal saline to dilute, add one-third of the diluent’s volume of Ficoll separation solution, centrifuge at 700g for 20-30min, reduce the lifting speed to zero, and absorb the middle buffy coat.

[0057] 1.2 Wash the buffy coat obtained in 1.1 twice with normal saline. According to the counting results, the inoculation density is 1×10 6 cell / ml, inoculate into RPMI1640 medium containing 10% FBS, add 1000UI / ml interferon-γ, add 300UI / ml interleukin-2 and 300UI / ml CD3 monoclonal antibody after 24h; add fluid on the 4th day, fluid density 5~10×10 5 cell / ml, and add 300 UI / ml of interleukin 2 according to the volume of rehydration; follow-up rehydration once every 3 days, and the density after rehydration is maintained at 1-1.5×10 6 cell / ml, and add 300UI / ml of interleukin 2 according to the volume of rehydration; culture for 12 to 14 days to obtain CIK cells.

[0058] 1.3 Pour out the CIK c...

Embodiment 2

[0064] 2.1 Pour out the CIK cells obtained in Example 1 from the culture flask, centrifuge at 400g for 5min, discard the supernatant and wash 2 times with PBS. After discarding the PBS, add the cell freezing solution (see the formula for the cell freezing solution) Table 2) Resuspended cells at a cell density of 3×10 7 cell / ml; take a part of the cell suspension for trypan blue staining to calculate the activity, and at the same time detect the surface markers CD3 and CD56 of CIK by flow cytometry; add the remaining cell suspension to cryopreservation tubes, divide each tube into 10 Day, 30 days, and 60 days in three groups, with 3 tubes of cells in each group, a total of 9 tubes of cells; the cryopreservation tubes were stored in a -80°C refrigerator, and transferred to liquid nitrogen for storage after 24 hours.

[0065] 2.2 Recovery of immune cells: Take out the immune cells stored in liquid nitrogen for 10 days, 30 days, and 60 days, and thaw them in a water bath at 37°C. ...

Embodiment 3

[0069] 3.1 Pour out the CIK cells obtained in Example 1 from the culture flask, centrifuge at 400g for 5min, discard the supernatant and wash with PBS twice, after discarding the PBS, add the cell freezing solution (see the formula for the cell freezing solution) Table 3) Resuspended cells at a cell density of 3×10 7 cell / ml; take a part of the cell suspension for trypan blue staining to calculate the activity, and at the same time detect the surface markers CD3 and CD56 of CIK by flow cytometry; add the remaining cell suspension to cryopreservation tubes, divide each tube into 10 Day, 30 days, and 60 days in three groups, with 3 tubes of cells in each group, a total of 9 tubes of cells; the cryopreservation tubes were stored in a -80°C refrigerator, and transferred to liquid nitrogen for storage after 24 hours.

[0070] 3.2 Recovery of immune cells: Take out the immune cells stored in liquid nitrogen for 10 days, 30 days, and 60 days, and thaw them in a water bath at 37°C. Th...

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Abstract

The invention provides a cell cryo-preserved liquid, an application, and an immune cell cryo-preservation method. The cell cryo-preserved liquid includes 0.7-0.9 ml / ml of a cell culture medium, 8-26 mg / ml of non-essential amino acids, 0.04-0.1 ml / ml of trehalose, 0.5-10 mg / ml of vitamin C, 10-50 mg / ml of human albumin, 0.04-0.12 ml / ml of propylene glycol, 0.01-0.07 ml / ml of dimethyl sulfoxide and 0.5-3 mg / ml of lentinan. Compared with a cell cryo-preserved liquid in the prior art, the human albumin can replace fetal calf serum to achieve the effects of the fetal calf serum in the cell cryo-preserved liquid, and meanwhile, the cell cryo-preserved liquid is added with the non-essential amino acids, the vitamin C and the lentinan, thereby ensuring cell proliferation vitality after recovery is not influenced. The trehalose and the propylene glycol can replace a protective effect of dimethyl sulfoxide, thereby ensuring moisture in cells to be not crystallized during approach of freezing point. The components are cooperated with each other to achieve cryo-preservation of immune cells.

Description

technical field [0001] The invention belongs to the technical field of cell engineering, and in particular relates to a cell cryopreservation solution, its application and an immune cell cryopreservation method. Background technique [0002] Immune cell therapy is a new type of autoimmune anti-cancer treatment. It uses biotechnology and biological agents to culture and amplify immune cells collected from patients in vitro and then reinfuse them back into patients to stimulate and enhance immunity. The body's own immune function, so as to achieve the purpose of treating tumors. [0003] With the rapid development of immune cell biology and immune molecular biology, somatic cell immunotherapy has become one of the important means of adjuvant therapy after radiotherapy and chemotherapy for tumor patients. All have good effects. [0004] However, the effect of in vitro culture of immune cells depends largely on the number and quality of immune cells in the patient's body, but ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 陈海佳王一飞葛啸虎万桦王小燕
Owner GUANGZHOU SALIAI STEMCELL SCI & TECH CO LTD
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