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Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof

A cryopreservation solution and cell suspension technology, applied in the field of cryopreservation solution, can solve the problems of unfavorable clinical application, long CIK cell culture cycle, easy contamination, etc., to expand blood volume, reduce the formation of ice crystals, and reduce cell swelling. Effect

Inactive Publication Date: 2013-07-24
XINXIANG MEDICAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

CIK cells have a long culture cycle and are prone to contamination, which is not conducive to their clinical application. Therefore, it is particularly important to find a way to preserve CIK cells

Method used

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  • Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof
  • Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof
  • Cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0026] Embodiment 1, the preparation of cell cryopreservation liquid

[0027] The preparation method of cell freezing liquid A: add dimethyl sulfoxide and dextran to the GT-T551 cell culture medium, so that the concentration of dimethyl sulfoxide is 10% (volume ratio), and the concentration of dextran 10% (volume ratio).

[0028] The preparation method of cell cryopreservation solution B: add dimethyl sulfoxide and dextran to the GT-T551 cell culture medium, so that the concentration of dimethyl sulfoxide is 5% (volume ratio), and the concentration of dextran 15% (volume ratio).

[0029] The preparation method of cell cryopreservation solution C: add dimethyl sulfoxide and dextran to GT-T551 cell culture medium, so that the concentration of dimethyl sulfoxide is 15% (volume ratio), so that the concentration of dextran 5% (volume ratio).

[0030] Cell cryopreservation solution D (traditional cell cryopreservation solution): add dimethyl sulfoxide and human serum albumin to G...

Embodiment 2

[0031] Embodiment 2, the acquisition and cryopreservation of CIK cells

[0032] 1. Acquisition of CIK cells

[0033] Umbilical cord blood: Obtained from full-term healthy fetuses of Xinxiang Medical College, with the consent of the family members.

[0034] 1. Use Ficoll lymphocyte separation medium (density gradient centrifugation) to separate mononuclear cells from cord blood.

[0035] 2. Put the mononuclear cells obtained in step 1 in 100ml of GT-T551 cell culture medium containing 1000μg / ml IFN-γ and 1% (volume ratio) autologous plasma (that is, umbilical cord blood plasma used to separate nuclear cells) at 37°C Let stand for 24 hours.

[0036] 3. Add CD3 monoclonal antibody and cytokine IL-2 to the culture system completed in step 2, so that the concentration of CD3 monoclonal antibody is 75ng / ml, and the concentration of cytokine IL-2 is 1000U / ml, and culture at 37°C for 24 Hour.

[0037] 4. Add 300ml of GT-T551 cell culture medium containing 1000U / ml cytokine IL-2 to...

Embodiment 3

[0071] Embodiment 3, acquisition and cryopreservation of CIK cells

[0072] The CIK cells used in this example were purchased from Shanghai Baili Biological Serum Network.

[0073] 1. Cryopreservation of CIK cells

[0074] Same as step 2 of embodiment 2.

[0075] 2. Recovery of CIK cells

[0076] 1. Same as step 3 of embodiment 2.

[0077] 2. Inoculate the CIK cells obtained in step 1 into GT-T551 cell culture medium containing 1000U / ml cytokine IL-2 and 1% (volume ratio) human plasma, and culture at 37°C.

[0078] 3. Morphological observation

[0079] 1. In step 2 of 2, start timing from inoculation of CIK cells, and collect CIK cells (that is, cells after thawing) 24 hours later.

[0080] 2. Observe the CIK cells before cryopreservation and the recovered cells under an inverted microscope, see Figure 5 .

[0081] Figure 5 Among them, D is the cells before cryopreservation, E is the cells of the experimental group A after recovery, and F is the cells of the control ...

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Abstract

The invention discloses a cryopreservation solution for preserving congenital intrathoracic kidney (CIK) cells and application thereof. The cryopreservation solution for preserving the CIK cells is prepared according to the following methods: adding dimethyl sulfoxide and dextran into a lymphocyte serum-free medium, so that the volume percent content of the dimethyl sulfoxide is 5-15 percent, and the volume percent content of the dextran is 5-15 percent. Compared with the cells before cryopreservation, the cells cryopreserved by employing the cryopreservation solution have the following advantages: (1) the cellular morphology does not have obvious difference; (2) immunophenotype (CD3+CD56+CD8+) does not have obvious difference; and (3) the cell proliferation activity and cell killing activity do not have obvious difference.

Description

technical field [0001] The invention relates to a cryopreservation solution for preserving CIK cells and application thereof. Background technique [0002] CIK cells, namely cytokine-induced killer cells (cytokine-induced killer cells), are non-MHC-restricted high-efficiency tumor-lytic cytotoxic T lymphocytes, and the proportion of peripheral blood lymphocytes is 1%-5%. In 1991, Schmidt-Wolf et al first reported a class of killer cells induced by various cytokines, namely CIK cells. [0003] At present, immune cell adoptive therapy has become one of the important means of adjuvant therapy for tumor patients after radiotherapy and chemotherapy, and CIK cells are the main force of adoptive therapy. Usually, autologous peripheral blood is used to prepare CIK. On the one hand, the number of CIK cells is insufficient, and on the other hand, the patient’s physical weakness will be aggravated, so that the body is in a period of weak immune system and is prone to infection. Umbil...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): A01N1/02
Inventor 官立萍姬明丽千智斌李振想武文杰李霞飞
Owner XINXIANG MEDICAL UNIV
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