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CIK cell, as well as preparation method and cell preparation thereof

A cell and IL-2 technology, applied in biochemical equipment and methods, drug combinations, microorganisms, etc., can solve the problems of low proportion and low proliferation of CIK cells, and achieve prolong life cycle, enhance tolerance, and resist The effect of broad tumor spectrum

Inactive Publication Date: 2009-09-02
上海德嘉生物科技有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

The proliferation of CIK cells by these methods is low, and the proportion of CD3+CD56+ cells and CD8+ cells in CIK cells is not high, and the therapeutic effect on tumors needs to be further improved

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0018] Embodiment 1 Preparation of CIK cells of the present invention

[0019] (1) Collection and preparation of peripheral blood mononuclear cells (PBMC)

[0020] Use a blood cell separator to collect anticoagulant blood from tumor patients under sterile conditions, double-dilution with normal saline, and add lymphocyte separation medium with a specific gravity of 1.077 (product of Tianjin Institute of Blood, Chinese Academy of Sciences Biotechnology Company) and diluted blood at a ratio of 1:2 and add to the centrifuge. In the tube, centrifuge at 2500rpm / 30min to separate PBMC, wash twice with Hanks solution, adjust the cell concentration with serum-free culture medium GT-T503, so that the cell concentration is 2×10 6 cells / ml of cell suspension.

[0021] (2) Novel human CIK cell induction and expansion of the present invention

[0022] Place the isolated cell suspension in GT-T503 culture medium containing PHA (phytohemagglutinin) 10U / ml, and put it into a 225cm 2 In the...

Embodiment 2

[0026] Example 2 Detection of various indicators of CIK cells

[0027] 1) Sterility test and heat source detection

[0028] For the CIK cells harvested in Example 1 and Comparative Example 1, both the sterility test and the heat source test were negative.

[0029] 2) Phenotype detection

[0030] Cell phenotype was detected by flow cytometry.

[0031] According to the phenotype detection results, in the CIK cells harvested in Example 1, T lymphocytes (CD3+ cells) accounted for more than 90% of the total number of cells. Among them, the proportion of each subset of T lymphocytes has a certain range of variation due to individual differences: the proportion of CD3+CD56+ cells is 40-60%, the proportion of CD8+ cells is 65-85%, and the proportion of CD3-CD56+ cells is 25-85%. 45%. Among the CIK cells harvested in Comparative Example 1, the proportion of CD3+CD56+ cells was 25-35%; the proportion of CD8+ cells was 45-60%.

[0032] 3) Viable cell count detection

[0033] The de...

Embodiment 3

[0038] Embodiment 3 Novel CIK cell preparation of the present invention

[0039] Put the CIK cells harvested after 10 days of in vitro culture in Example 1 into a 50ml centrifuge tube, centrifuge at 1000rpm / 10min, and collect 1×10 10 Wash the cells once with normal saline, discard the supernatant, beat the precipitated cells with normal saline, transfer to a 100ml infusion bottle, add 500,000 units of IL-2, 100ml of normal saline for injection, prepare a cell suspension, and keep the sample , for indicator detection. The CIK cell viability count is greater than 95%, and the bacteria test and heat source test are all negative. The qualified ones are covered and sealed, and stored at low temperature for later use.

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PUM

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Abstract

The invention discloses a CIK cell, as well as a preparation method and a cell preparation thereof. The method for preparing the CIK cell comprises the following steps: placing a separated mononuclear cell in a culture fluid containing phytohemagglutinin; transplanting the mononuclear cell into a culture flask enveloped by antiCD3 monoclonal antibody and antiCD28 monoclonal antibody after the cell is cultured for 24 to 72 hours; and adding a culture fluid containing IL-1alpha and IL-2 into the culture flask to keep on culturing for 5 to 15 days, and separating the cells into different flasks to be cultured every 2 to 3 days. The CIK cell prepared by the method has the characteristics of obvious improved cell proliferation, great increase of CD8 cell proportion, wide antineoplastic spectrum and strengthened antineoplastic activity. The CIK cell and the cell preparation can effectively prevent the metastasis and the recrudescence for of postoperative patients with tumor, can be combined with chemicotherapy to effectively reduce toxic and side effects of the chemicotherapy, strengthens the survivability tolerance of the patients to improve healing efficacy, and can obviously prolong lifecycle to of end-stage patients so as to improve the life living quality.

Description

technical field [0001] The invention belongs to the field of in vitro cell culture, and in particular relates to a CIK cell, a preparation method and a cell preparation thereof. Background technique [0002] Tumor biotherapy is the fourth treatment mode after surgery, radiotherapy, and chemotherapy. CIK cell (Cytokine induced killer cell, CIK) therapy is currently the most effective tumor biotherapy method recognized internationally. [0003] CIK cells are a group of heterogeneous cells obtained after co-cultivating human peripheral blood mononuclear cells with various cytokines (such as anti-CD3 McAb, IL-2, IFN-γ, IL-1, etc.) for a period of time under in vitro conditions. Because its main effector cells have both T cell surface markers (TCR-α / β, CD3) and NK cell surface markers (CD56), it has both the strong anti-tumor activity of T lymphocytes and the non-MHC of NK cells. (Major Histocompatibility Complex) Restricted Tumoricidal Advantage. The main effector cells in CIK...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08A61K35/14A61P35/00A61K35/17
Inventor 徐鸣
Owner 上海德嘉生物科技有限公司
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