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Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells

A cryopreservation method and cryopreservation technology, applied in the field of biomedicine, can solve the problems of low cytotoxic activity, long cost cycle, waste of medical costs, etc., and achieve the effects of simplifying the process, improving the permeability, and omitting steps.

Inactive Publication Date: 2016-02-24
RUIAN PULUO BIOTECH
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, in the preparation technology of CIK cells in the prior art, there are disadvantages of poor proliferative ability and low cytotoxic activity of CIK cells
Moreover, it takes a long time to induce CIK cells, and multiple blood collections are required for clinical induction, which causes great pain to patients and wastes a lot of medical costs.

Method used

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  • Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells
  • Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells
  • Cryopreservation method and medium for CIK (Cytokine Induced Killer) cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0030] The cryopreservation of embodiment 1CIK cell

[0031] Cryopreservation and recovery of CIK cells includes the following steps:

[0032] 1. Cryopreservation of CIK cells

[0033] a. Take CIK cells (CIK cells can be prepared by conventional methods in this field, for example, refer to the method in Chinese patent CN201210324368.4), centrifuge at 1000rpm for 5min, take out the supernatant and freeze it at -80°C for later use.

[0034] b. Use physiological saline to prepare freezing storage medium according to the following table

[0035]

[0036]

[0037] c. CIK cells obtained by centrifugation were placed in freezing medium, reduced to 30-60% of the volume of the original CIK culture medium, frozen at -20°C for 3 hours, then frozen in a -80°C refrigerator for 8 hours, and then frozen in liquid nitrogen live.

[0038] 2. Recovery of CIK cells

[0039] d. Take out the supernatant frozen in step a and thaw it in a refrigerator at 4° C., then centrifuge the supernat...

Embodiment 2

[0042] Example 2: Detection of CIK cells

[0043] 1. CIK cell live cell detection

[0044] Take 100ul of recovered CIK cells, add 100ul of 0.4% placenta blue staining solution, live cells will not be stained, and dead cells will be stained blue. Experimental results such as figure 1 As shown, it can be seen from the figure that the cryopreservation medium of the present invention can make the viable cell rate of the resuscitated cells reach 99%, but the pH of the cryopreservation medium has a great influence on the cell resuscitation, when the pH is as low as 6 or when the pH When it is higher than 6.5, the viable cell rate is significantly reduced.

[0045] 2. Detection of CIK cytotoxic activity

[0046] The CIK cells of each group revived in Example 1 were added to the 96-well plate of the K562 tumor cell line (purchased from Wuhan Punuosai Life Technology Co., Ltd.) cells inoculated for 24 hours according to the effect-to-target ratio of 10:1, and after 24 hours of co-cu...

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Abstract

The invention discloses a cryopreservation method and medium for CIK (Cytokine Induced Killer) cells. Concretely, the cryopreservation method for the CIK cells comprises the steps: centrifuging a prepared CIK cell culture solution for 3-5 minutes at the rate of 800-1,500rpm, and cryopreserving supernatant at the temperature of -80 DEG C for later use; and putting CIK cells obtained through centrifugation into the cryopreservation medium, carrying out freezing for 2-4 hours at the temperature of -20 DEG C, then, carrying out freezing for 6-10 hours at the temperature of -80 DEG C, and then, storing the frozen CIK cells in liquid nitrogen, wherein the cryopreservation medium contains 30-100 micrograms / milliliter of human serum albumin, 1-5 micrograms / milliliter of vitamin E, 20-50 micrograms / milliliter of glutathione and 10-20 micrograms / milliliter of glycine. The cells cryopreserved by using the cryopreservation medium are high in recovery ratio, and characteristics such as cytotoxic activity of the CIK cells are maintained after recovery. All ingredients of the cryopreservation medium do not affect subsequent clinical cell therapy application.

Description

technical field [0001] The invention relates to the field of biomedicine, in particular to a CIK cell cryopreservation method and a CIK cell cryopreservation medium. Background technique [0002] Cytokine-induced killer cells (cytokine-induced killer cells, CIK cells) are a group of heterogeneous cells obtained from human peripheral blood, umbilical cord blood or bone marrow mononuclear cells cultured with cytokines for a period of time in vitro. Years of clinical research at home and abroad have shown that CIK cell therapy can improve the disease-free survival and overall survival of patients, improve the body's anti-tumor immune function, and improve the quality of life of patients. Because this kind of cell expresses two kinds of membrane protein molecules, CD3 and CD56, it is also called NK cell-like T lymphocyte, which has both the strong anti-tumor activity of T lymphocytes and the non-major histocompatibility complex of NK cells (majorhistocompatibility complex, MHC)...

Claims

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Application Information

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IPC IPC(8): A01N1/02C12N5/0783
Inventor 杨钟华
Owner RUIAN PULUO BIOTECH
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