Natural killer cell culture substrate and amplification culture method for natural killer cells

A natural killer cell and culture substrate technology, which is applied in the field of natural killer cell culture substrate and natural killer cell expansion and culture, can solve the problem that the safety has yet to be discussed, it is difficult to meet large-scale experiments, clinical tumor immunotherapy, high cell purity, etc. problems, to achieve the effect of enhancing cell killing activity, alleviating the cumbersome production process, and enhancing cytotoxicity

Active Publication Date: 2017-12-15
TIANJIN CHANGHE BIOLOGICAL TECH
View PDF12 Cites 16 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0007] However, the above-mentioned culture methods have a certain degree of disadvantages. Among them, the method of using virus-transfected B lymphocytes and tumor cells as feeder cells to increase the amplification factor of natural killer cells is cumbersome and the safety has yet to be explored; Using the negative sorting method of immunomagnetic bead sorting system for natural killer cell culture, the production

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Natural killer cell culture substrate and amplification culture method for natural killer cells
  • Natural killer cell culture substrate and amplification culture method for natural killer cells
  • Natural killer cell culture substrate and amplification culture method for natural killer cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0068] Example 1: Lymactin-NK antibody coating

[0069] The Lymactin-NK antibody with a concentration of 0.6mg / mL was coated on the cell culture flask, the coating condition was 37°C, and the incubation time was 2h to obtain the coated cell culture flask.

Embodiment 2

[0070] Example 2: Preparation of autologous plasma and human peripheral blood mononuclear cells

[0071] Collect peripheral blood, transfer the collected peripheral blood to a 50mL centrifuge tube, and centrifuge at 2000rmp, 20°C for 10 minutes to obtain autologous plasma; then use lymphocyte separation medium to separate and collect human peripheral blood mononuclear cells. The collected human peripheral blood mononuclear cells were washed three times with PBS buffer to obtain human peripheral blood mononuclear cells.

[0072] Wherein, the steps of separating and collecting human-derived peripheral blood mononuclear cells using lymphocyte separation medium specifically include:

[0073] 1. Add the blood sample after drawing the plasma into PBS at a ratio of 1:1, and mix well;

[0074] 2. Slowly add the diluted blood sample on the surface of the lymphocyte separation liquid, and the ratio of the diluted blood sample to the lymphocyte separation liquid is 1:1;

[0075] 3. Cen...

Embodiment 3

[0077] Embodiment 3: induction culture

[0078] Step 1: Use X-VIVO 15:AlyS505NK-AC=1:1 serum-free medium to resuspend the human peripheral blood mononuclear cells obtained in Example 2, and adjust the cell concentration in the medium to 1.5×10 6 / mL, then inoculated into the cell culture flask coated with Lymactin-NK antibody obtained in Example 1;

[0079] Step 2: Add the autologous plasma obtained in Example 2 into the cell culture flask of step 1 above, the concentration of the autologous plasma in the natural killer cell culture medium is 5%, and add cytokine: IL-2 (1000IU / mL), IL-12 (5ng / mL), IL-15 (20ng / mL), IL-21 (10ng / mL), followed by induction culture for 14 days;

[0080] Step 3: In the first 7 days of the induction culture process, perform rehydration every 3 days, and use the rehydration medium to adjust the cell density to 1.5×10 6 / mL, and supplemented with autologous plasma and IL-2, IL-12, IL-15, IL-21 cytokines; in the last 7 days of the induction culture pr...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

PUM

No PUM Login to view more

Abstract

The invention provides a natural killer cell culture substrate and an amplification culture method for natural killer cells, and relates to the technical field of cell culture. The natural killer cell culture substrate is mainly composed of a serum-free culture medium, autologous plasma and cytokines; according to the amplification culture method for the natural killer cells with the natural killer cell culture substrate, the IL-2, IL-12, IL-15, IL-21 cytokines in the cell culture substrate are combined through Lymactin-NK antibodies to induce mononuclear cells of human peripheral blood to release dangerous signals, the natural killer cells are activated, and the killing activity of the cells is enhanced. According to the method, a large quantity of high-quality natural killer cells can be obtained in a short period through efficient amplification, and the problems are effectively solved that in existing culture methods for the natural killer cells, the production process is complex, the production cost is relatively high, and natural killer cells are low in amplification multiple, not high in purity and difficult to produce in a large-scale mode.

Description

technical field [0001] The invention relates to the technical field of cell culture, in particular to a natural killer cell culture substrate and a natural killer cell expansion culture method. Background technique [0002] Natural killer cells (Nature Killer cells, NK) are cytotoxic lymphocytes of the body's innate immune system, which exist in lymphoid organs and peripheral tissues, and have functions such as anti-tumor, anti-infection, and immune regulation. Direct recognition and non-specific killing of tumor cells is the first barrier of the human defense system. Natural killer cells have strong cytotoxic activity, they respond very quickly to stimulating factors, and have a high intensity of immune response; at the same time, the killing activity of natural killer cells does not require antigen stimulation and is not limited by MHC molecules; in addition, natural killer cells The cells also have a strong cytokine / chemokine secretion function, which helps to initiate a...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to view more

Application Information

Patent Timeline
no application Login to view more
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/2312C12N2501/2315C12N2501/2321
Inventor 徐永胜李伟李政楠
Owner TIANJIN CHANGHE BIOLOGICAL TECH
Who we serve
  • R&D Engineer
  • R&D Manager
  • IP Professional
Why Eureka
  • Industry Leading Data Capabilities
  • Powerful AI technology
  • Patent DNA Extraction
Social media
Try Eureka
PatSnap group products

Browse by: Latest US Patents, China's latest patents, Technical Efficacy Thesaurus, Application Domain, Technology Topic.

© 2024 PatSnap. All rights reserved.Legal|Privacy policy|Modern Slavery Act Transparency Statement|Sitemap