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Efficient CIK amplifying method

A high-efficiency, IL-2 technology, applied in the field of cell therapy, can solve the problems of low CD3+CD56+ double positive expression rate and low CIK cell proliferation rate

Active Publication Date: 2015-11-25
UNION STEMCELL & GENE ENG
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] The purpose of the present invention is to provide a method for efficiently amplifying CIK, which can significantly improve the CIK cell proliferation multiple and cytotoxic activity, in order to solve the problems of low multiplication factor and low CD3+CD56+ double-positive expression rate of CIK cells obtained by the existing method. The multiple of expanded cells is more than 1000 times, and the expression rate of CD3+CD56+ double positive is more than 30%.

Method used

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Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0044] Aseptically collect 100ml of peripheral blood from healthy people or patients, transport it to the immune cell therapy room within 2 hours, and separate PBMC and in vitro culture of CIK in the clean workshop. Whole blood can be separated and cultured after passing the microbial immunoassay.

[0045] PBMC isolation:

[0046] ●Peripheral blood and normal saline were mixed in equal volume, and left at room temperature for 5 minutes.

[0047] ●Take 200ul and count with a blood cell counter.

[0048] ●80ml Ficoll centrifuged to separate PBMC.

[0049] ●Centrifugal parameters: 500g, 19 degrees, 20 minutes.

[0050] ●Take the white film.

[0051] ●Add physiological saline to a total volume of 100ml, wash twice.

[0052] ●Centrifugal parameters: 300g, 19 degrees, 5 minutes.

[0053] ●Use a pipette or a vacuum pump to remove the supernatant, do not pour. The remainder of the supernatant was 0.5-1 cm above the cell pellet.

[0054] ●The cells were resuspended in 10ml lympho...

Embodiment 2

[0058] CIK induction culture:

[0059] ●After counting the isolated PBMC, press 5×10 5 Individual / ml density inoculation.

[0060] ● Add the cells into T175 culture flasks coated with CD3 mAb (10 ng / ml) and CD28 mAb (50 ng / ml).

[0061] ●Supplement complete lymphocyte medium to 100ml, add IL-2 (500IU / ml).

[0062] ●The next day, add IFN-γ (701IU / ml) and IL-1α (51IU / ml) to the culture flask respectively.

[0063] ● On day 3, count the cells and add IL-2 (500 IU / ml).

[0064] ● On day 5, count the cells and add 100ml of lymphocyte complete medium and IL-2 (500IU / ml).

[0065] ● On day 7, count the cells, transfer the cell suspension into a cell culture bag, add 200ml complete medium for lymphocytes, CD3mAb (10ng / ml), CD28mAb (50ng / ml), IL-2 (500IU / ml).

[0066] ●Add lymphocyte complete medium and IL-2 (500IU / ml) with the same volume as the original culture system every 2 days.

[0067] ● On day 13 and day 16, cells were counted and cells were harvested.

[0068] Remarks: ...

Embodiment 3

[0075] CIK induction culture:

[0076] After counting the isolated PBMCs, press 7×10 5 Individual / ml density inoculation.

[0077] ● Add the cells into T175 culture flasks coated with CD3mAb (20ng / ml) and CD28mAb (70ng / ml).

[0078] ●Supplement complete lymphocyte medium to 100ml, add IL-2 (700IU / ml).

[0079] ●The next day, add IFN-γ (800IU / ml) and IL-1α (70IU / ml) to the culture flask respectively.

[0080] ● On day 3, count the cells and add IL-2 (700 IU / ml).

[0081] ● On day 5, count the cells and add 100ml of lymphocyte complete medium and IL-2 (700IU / ml).

[0082] ● On day 7, count the cells, transfer the cell suspension into the cell culture bag, add 200ml complete medium for lymphocytes, CD3mAb (20ng / ml), CD28mAb (70ng / ml), IL-2 (700IU / ml).

[0083] ●Add lymphocyte complete medium and IL-2 (700IU / ml) equal to the volume of the original culture system every 2 days.

[0084] ● On day 13 and day 16, cells were counted and cells were harvested.

[0085] Remarks: The...

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Abstract

The invention discloses an efficient CIK amplifying method, in particular to a method for cell populations, namely cytokine-induced killer cells utilizing in-vitro cell factors for efficiently inducing mononuclear cell expressions CD3 and CD56 of peripheral blood, wherein the cell populations have killing functions. The cultivation method for the CIK efficiently expressing CD3+CD56 comprises the following steps that the peripheral blood of a healthy person or a patient is collected in a sterile mode, after the peripheral blood is diluted through a saline solution with the same volume, a Ficoll lymphocyte separating medium is used for separating mononuclear cells, in the CIK cell inducing process, CD3 monoclonal antibodies (CD3mAb), CD28 monoclonal antibodies (CD28mAb), interferon-gamma (IFN-gamma), interleukin-2(IL-2) and interleukin 1alpha (IL-1alpha) are added, cultivation is carried out for 13-16 days to obtain cells, a CIK cell preparation is prepared, and flow cytometry detection and microorganism detection are carried out. According to the CIK cultivation method, the CIK number can be increased to be 6*10<9> or over 6*10<9> in two weeks, the cell survival rate can be increased to be 99% or over 99%, and the double-positive proportion of the CD3+CD56+cells reaches 30% or over 30%.

Description

technical field [0001] The invention belongs to the field of cell therapy, and in particular relates to a method for efficiently amplifying CIK. Background technique [0002] In recent years, the incidence and mortality of tumors have been increasing year by year in my country. According to the "2012 China Cancer Registration Annual Report", there are about 3.5 million new cancer cases in my country every year, and about 2.5 million cancer deaths. Cancer patients, and the incidence of cancer tends to be younger. Adoptive immune cell therapy is an emerging new anti-tumor treatment method with significant curative effect in tumor rehabilitation medicine. It is another treatment method after the three traditional treatment methods of surgery, radiotherapy and chemotherapy. The biggest advantage lies in its individuality, safety, targeting and efficiency. [0003] Cytokine-Induced Killer cells (CIK) is a kind of immune cell therapy, which was first reported in 1991 by biologist...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
Inventor 陈晓波洪敬欣韩洪起韩俊领武文杰靳霞苏相相郝世凯张宇光王雪莲
Owner UNION STEMCELL & GENE ENG
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