Culture composition for amplifying primary NK cells in vitro without feeder layer and application thereof

A technology of NK cells and compositions, which is applied in the field of compositions for cultivating NK cells, can solve the problems that NK technology products cannot be widely used, safety risks of feeder layer cells, and cumbersome operation steps, so as to achieve optimal technology selection and reduce T cell Ratio, the effect of improving the preparation efficiency

Active Publication Date: 2018-10-09
北京亦科诺生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, on the one hand, due to the high preparation cost and cumbersome operation steps of the above-mentioned technology; on the other hand, due to the safety risk of the clinical application of feeder cells, the current ...

Method used

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  • Culture composition for amplifying primary NK cells in vitro without feeder layer and application thereof
  • Culture composition for amplifying primary NK cells in vitro without feeder layer and application thereof
  • Culture composition for amplifying primary NK cells in vitro without feeder layer and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0057] The preparation of embodiment 1.NK culture medium

[0058] First configure the medium for NK activation and expansion. Activation medium (AM) and expansion medium (Proliferation medium, PM) are both based on basal medium, and are obtained by adding different concentrations of cytokines, recombinant proteins and small molecules. The specific preparation components are shown in the table below 1 (In the NK activation medium, Anti-CD16mAb and Anti-CD52mAb can be added with one of the components).

[0059] Table 1. Preparation of NK preparation medium

[0060]

[0061] The main preparation method is:

[0062] 1) Dissolve the lyophilized powder of recombinant human IL-2 (rhIL-2), IL-21 (rhIL-21) and IL-15 (rhIL-15) in sterile ultrapure water, according to the final concentration in the culture system, Prepared as a 1000000IU / mL stock solution, aliquoted and stored in a -80°C refrigerator until use.

[0063] 2) The OK432 lyophilized powder was dissolved in physiologica...

Embodiment 2

[0067] Embodiment 2. Utilize peripheral blood PBMC to carry out NK preparation

[0068] Peripheral blood mononuclear cells (PBMC) were obtained by density gradient separation of 35 mL of fresh anticoagulated peripheral blood according to conventional methods. The isolated PBMCs were grouped according to Table 2 below.

[0069] Table 2. Experimental grouping

[0070] Numbering

group

Grouping and Processing

1

Experimental group 1

Unpacked activated + NK-PM

2

Experimental group 2

Not packaged to be activated + NK-PM (including DST)

3

Experimental group 3

Package is activated +NK-PM

4

Experimental group 4

Package is activated + NK-PM (including DST)

5

positive control

feeder culture

[0071] Groups 1 and 2 were resuspended in NK-AM medium containing 5% autologous serum (basal medium was X-VIVO10, Anti-CD16mAb was selected for both Anti-CD16mAb and Anti-CD52mAb), and the cell density was ad...

Embodiment 3

[0077] Example 3. Utilization of PBMCs derived from umbilical cord blood for NK preparation

[0078] PBMCs were isolated from freshly collected umbilical cord blood by density gradient centrifugation. After separation, resuspend the cells in erythrocyte lysate and mix gently to remove residual erythrocytes in PBMCs. The treated PBMCs were grouped according to the grouping method in Example 2, and NK was prepared, and the preparation period was 14 days. The basal medium of the NK-AM medium used in the preparation is SCGM; the basal medium of the NK-PM medium is X-VIVO15. After the preparation, the total number of cells in each experimental group, the purity of NK and the proportion of T cells in the final product were detected by flow cytometry.

[0079] The analysis results show that the preparation of NK by using the PBMC derived from umbilical cord blood using the technology of the present invention obtains similar results to those in Example 1, and the uncoated activation...

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Abstract

The invention provides a culture composition for amplifying primary NK cells in vitro without a feeder layer and the application thereof, and relates to obtained NK cells and related application. Theinvention provides a composition for culturing the NK cells. The composition comprises an activating culture medium composition and/or an amplifying culture medium composition, wherein the activatingculture medium composition comprises an activating basal culture medium, rhIL-2, rhIL-15, rhIL-21, OK432, OKT-3 and Anti-CD16mAb or Anti-CD52mAb; the amplifying culture medium composition comprises anamplifying basal culture medium, rhIL-2, rhIL-15, rhIL-21 and DST. Through the adoption of the technology provided by the invention, the in-vitro preparation efficiency of NK is improved and the preparation cost is reduced; the NK purity of a final product is increased, and the obtained NK cells have higher biological activity.

Description

technical field [0001] The present invention relates to a composition for cultivating NK cells and related methods, in particular to a composition for culturing primary NK cells in vitro without a feeder layer and its application, as well as the obtained NK cells and related applications. Background technique [0002] Natural killer cell (NK) is a kind of CD3 cell in the human immune system - / CD56 + CD56-high expression (CD3 - / CD56 bright ) and low expression of CD56 (CD3 - / CD56 dim ) two subgroups. NK cells widely exist in tissues and organs including peripheral blood, skin and mucous membranes, and liver; they play an important role in the body's anti-infection, anti-virus and anti-tumor immune responses. [0003] With the breakthrough development of immune cell anti-tumor therapy technology, the immunological activity of NK, especially its anti-tumor-related biological activity has received more and more attention. A large number of studies have reported in deta...

Claims

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Application Information

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IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/72C12N2501/2302C12N2501/2315C12N2501/2321C12N2501/515C12N2501/599
Inventor 不公告发明人
Owner 北京亦科诺生物科技有限公司
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