CIK (cytokine-induced killer) frozen stock solution and frozen preservation method

A cryopreservation method and technique for cryopreservation, which are applied in the preservation, application, and animal husbandry of human or animal bodies, can solve the problems of complicated cryopreservation procedures, reduced activity, and difficulty in obtaining serum autologous serum, and achieve simplified cryopreservation. The effect of saving method and improving the survival rate

Active Publication Date: 2015-09-30
ADLAI NORTYE BIOPHARMA CO LTD
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Therefore, the serum-free cryopreservation solution reduces the activity of frozen cells after recovery, which cannot achieve our goal of efficient value-added.
However, the use of serum is difficult to obtain autologous serum, and exogenous serum has the risk of infectious diseases
Moreover, the existing cryopreservation procedures are complicated, which is not conducive to the actual operation.

Method used

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  • CIK (cytokine-induced killer) frozen stock solution and frozen preservation method
  • CIK (cytokine-induced killer) frozen stock solution and frozen preservation method

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0066] Example 1: Preparation of logarithmic phase CIK cells

[0067] (1) Separate peripheral blood mononuclear cells from peripheral blood, resuspend in X-VIVO15 serum-free medium, and make the cell concentration of peripheral blood mononuclear cells 1×10 6 cells / mL, add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, static culture for 3 days;

[0068] (2) Add X-VIVO15 serum-free medium to 100 mL in the culture medium prepared in step (1), and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, static culture for 1 day;

[0069] (3) Add X-VIVO15 serum-free medium to 200-240mL to the culture medium prepared in step (2), and add IL-2 at the same time, so that the concentration of IL-2 is 1×10 3 U / mL, static culture for 3 days; seed CIK cells in the logarithmic growth phase were prepared.

Embodiment 2

[0070] Embodiment 2: the preparation of CIK cell cryopreservation liquid

[0071] (1) Place human autologous plasma in a sterile centrifuge tube and centrifuge at 1800rpm for 6 minutes;

[0072] (2) Absorb the upper layer of plasma, transfer it to a new centrifuge tube, centrifuge at 3000rpm for 20 minutes, and absorb the supernatant, which is the human autologous plasma for cryopreservation;

[0073] (3) Mix dimethyl sulfoxide, the above-mentioned human autologous plasma, and medium X-VIVO15 at a volume ratio of 1:4:5 to obtain CIK cell cryopreservation solution.

Embodiment 3

[0074] Example 3: Cryopreservation of CIK cells

[0075] (1) Take the CIK cells cultured to the 7th day, centrifuge at 1800rpm for 6 minutes, discard the supernatant, add the above CIK cell freezing solution, and adjust the cell concentration to 1*10 8 Each / mL, each / 5mL was added to a 5mL sterile cryopreservation tube. Place the cryovials in a programmed cooling box pre-cooled at 4°C;

[0076] (2) Place the programmed cooling box with the cryopreservation tube in a -80°C refrigerator for 24 hours, and then store the cryopreservation tube in liquid nitrogen for long-term storage.

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Abstract

The invention provides a frozen stock solution for CIK (cytokine-induced killer) cells and a frozen preservation method. According to the frozen stock solution, serum is replaced with autologous plasma of a user, so that the risk of an infectious disease in exogenous serum is avoided. The adopted reagent is suitable for a simple programmed cooling method of a programmed cooling box. The reagent provided by the invention contains dimethyl sulfoxide, autologous plasma and a culture medium at the ratio of 1 to 4 to 5. The frozen stock solution has the characteristics of simple method and high cell resuscitation activity, and is suitable for storage of immune cells by patients at an earlier stage of chemoradiotherapy or healthy people in a manner of the CIK cells for later CIK cell therapy.

Description

technical field [0001] The invention belongs to the technical field of cell cryopreservation, and in particular relates to a novel CIK cell cryopreservation solution and a cryopreservation method. The invention avoids the risk of infectious diseases caused by exogenous serum by adding human autologous plasma, and the reagents used are suitable for the simple programmed cooling method of the programmed cooling box. The invention has the characteristics of simple method and high cell recovery activity. Background technique [0002] CIK cells, Cytokine-Induced Killer (CIK) cells are a new type of immunocompetent cells. CIK cells have strong proliferation ability, strong cytotoxicity, and certain immune characteristics. Because the cells express two membrane protein molecules, CD3 and CD56, they are also called NK cell (natural killer cell)-like T lymphocytes, which have both the strong anti-tumor activity of T lymphocytes and the non-MHC restriction of NK cells. Tumor killing...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 何学松闫桂云杨东晖路杨
Owner ADLAI NORTYE BIOPHARMA CO LTD
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