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A method for cultivating self activated lymphocyte

A technology of lymphocytes and culture methods, applied in the direction of cell culture active agents, biochemical equipment and methods, animal cells, etc., can solve problems such as limitations, and achieve the effects of simple administration, improved prognosis, and less side effects

Inactive Publication Date: 2009-12-16
NKBIO
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] However, the above-mentioned method of activating CD4 T cells has the following technical problem; because this method can only selectively activate T cells involved in the acquisition of immunity among immune cells, when this method is used to treat tumors, although a large number of activated T cells can Effectively attack or kill cancer cells that have been memorized, but it is limited in the treatment of malignant tumors by attacking various cancer cells that have not been memorized

Method used

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  • A method for cultivating self activated lymphocyte
  • A method for cultivating self activated lymphocyte
  • A method for cultivating self activated lymphocyte

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Example 1: Preparation of Autoactivated Lymphocytes

[0045] Lymphocytes obtained from 60cc of the patient's peripheral blood were suspended in 3ml of culture medium, mixed in 33ml of culture medium supplemented with 70ul IL-2, 200ul L-glutamine and 3ml of self-derived plasma. In the presence of anti-CD3, anti-CD16 and anti-CD56, culture for 4 days (the first culture step), and then mix in 60ml of culture medium added with 100ulIL-2, 1ml of L-glutamine and 7ml of self-derived plasma , continue culturing for 3 days (the second culturing step), then add 7ml of plasma derived from itself to the culture mixture after the second culturing step, inject it into a gas-permeable culture bag containing 1L of culture fluid, and culture for 7 more days. days (the third cultivation step).

Embodiment 2

[0046] Example 2: Observing changes in phenotype and cell number before and after culture

[0047] figure 1 It is a schematic diagram of the phenotype changes of activated lymphocytes before and after culture. The H1 area represents NK cells, the H4 area represents T cells, the H2 area represents NKT cells, and the H3 area represents the distribution of other immune cells. figure 2 It is a schematic diagram of the change of the number of immune cells in the culture process according to the present invention.

[0048] The activated lymphocytes cultured by the same method as in Example 1 were analyzed by flow cytometry (floweytometry) for surface antigens, and figure 1 To illustrate the results, as shown in Figure (a), the distribution of surface antigens before culture is mainly in the H4 region, and as shown in Figure (b), the distribution of surface antigens after culture is mainly in the H1 region, and the actual calculation of CD3 positive (Positive ) T cells and CD16+...

Embodiment 3

[0050] Example 3: Analysis of Cytotoxicity to Various Cancer Cells

[0051] image 3 It is a schematic diagram of the cytotoxicity analysis results of the activated lymphocytes obtained according to the present invention.

[0052] When activated lymphocytes cultured by the method of Example 1 were used as effector cells, a blood cancer cell line (K562) was used as target cells, and the ratio of cancer cells to activated lymphocytes was set at 10:1, the activated lymphocytes were measured. Cytotoxicity analysis was performed on the killing ability of cells against blood cancer cell lines, and the results were as follows: image 3 As shown, the cytotoxicity of activated lymphocytes increased 6.8 to 16.4 times compared with lymphocytes isolated from normal blood.

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Abstract

Provided is a method of culturing self-activated lymphocytes applicable to the treatment of malignant tumors. The method raises the percentage's of natural killer (NK) cells of the lymphocytes and evenly activate the NK cells, T cells and natural killer T (NKT) cells, and thus can be used to effectively eliminate various kinds of cancer cells. The method of culturing self- activated lymphocytes involves: extracting lymphocytes from human peripheral blood; performing a first culturing step of culturing the extracted lymphocytes in a culture fluid to which IL-2, L-glutamine and autochthonous plasma are added, in the presence of anti-CD3, anti- CD 16, and anti-CD56 antibodies; and performing a second culturing step of culturing the mixed culture fluid resulting from the first culturing step in the presence of anti-CD3, anti-CD16, and anti-CD56 antibodies after being admixed with a culture fluid to which IL-2, L-glutamine and autochthonous plasma are added.

Description

technical field [0001] The present invention relates to a method for culturing autoactivated lymphocytes for the treatment of malignant tumors, more specifically, the present invention relates to the presence of interleukin 2 (IL-2) and anti-CD3, anti-CD16, and anti-CD56 antibodies Under certain conditions, culture lymphocytes isolated from human peripheral blood, increase the proportion of NK cells in lymphocytes, uniformly activate NK cells, T cells and NKT cells, and effectively remove a variety of cancer cell culture methods. Background technique [0002] The treatment methods for malignant tumors that occur in the human body generally include surgery, radiation therapy, and chemotherapy. However, malignancies may also exist in anatomical sites that are difficult to treat with these methods, in which case vigorous adjuvant therapy is necessary to improve patient outcomes. [0003] In adjuvant therapy, adoptive immunotherapy (adaptive immunotherapy) that activates immune...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/08C12N5/0783
CPCC12N2501/515C12N2501/23A61K39/0011C12N5/0646C12N5/0636A61K2039/5158C12N2501/599C12N5/0634A61K2239/48A61K39/4613A61K39/4644A61K39/4611C12N5/00
Inventor 李东旭洪藓旼崔炳仁
Owner NKBIO
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