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Culture medium for culturing immune cells isolated from umbilical cord blood and culture method thereof

A technology of immune cells and culture methods, applied in the field of improved immune cell culture medium, which can solve the problems of high cost, cell growth speed, cell activity, cell killing ability, etc., and achieve the effects of avoiding quality changes, fast growth speed, and high activity

Active Publication Date: 2020-03-03
上海中溢精准医疗科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0009] In view of the many defects in the commercial mass production of immune cells currently used clinically at home and abroad in adjuvant therapy and medical care, such as high cost, problems with cell growth speed, cell activity problems, and immune cells’ damage to tumor cells or aging The present invention provides a serum-free culture medium with an improved formula for the cultivation of immune cells isolated from umbilical cord blood

Method used

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  • Culture medium for culturing immune cells isolated from umbilical cord blood and culture method thereof
  • Culture medium for culturing immune cells isolated from umbilical cord blood and culture method thereof
  • Culture medium for culturing immune cells isolated from umbilical cord blood and culture method thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0036] The formulation of the GR medium for cultivating immune cells isolated from umbilical cord blood provided in this example is:

[0037] The basal medium is CTS AIM-V SFM medium containing KSR (Knockout serum replacement) serum substitute with a volume concentration of 20%, in which the following five cytokines are added:

[0038]

Embodiment 2

[0052] This embodiment provides a method for culturing immune cells induced by cytokines, so as to obtain a new type of immunocompetent cells. Specifically, it is cultivated and obtained by the following methods:

[0053] (1) Separation of PBMC:

[0054] ①In a 15mL centrifuge tube pre-added with 3mL Ficoll lymphocyte separation solution, use a 10mL syringe to combine with a 1mL syringe needle, slowly add 5mL of cord blood into the above centrifuge tube (inject along the wall, keep stratification) 400g, increase speed 9, decrease speed 0 , centrifuged for 30 minutes; use a 3mL sterile dropper to take out the upper plasma of the centrifugate, and transfer it to a 50mL centrifuge tube (all plasma is combined into the same centrifuge tube);

[0055] ②Use a 3mL sterile dropper to take the buffy coat and its upper layer of residual plasma and a small amount of Ficoll lymphocyte separation solution in the lower layer, and transfer them to a clean 50mL centrifuge tube (put the buffy ...

Embodiment 3 3

[0059] Embodiment 3 three kinds of medium culture effect comparisons

[0060] (1) Comparison of cell growth

[0061] Use GR, G, and 505 medium to culture immune cells according to the method in Example 2, and do three repeated experiments with three cord blood to verify whether the effect of the three mediums is reproducible, and exclude other conditions. interference. The trypan blue activity was determined when each experiment was carried out to the 13th day, and the data of the second experiment were taken as an example. The three media were operated in parallel, and the initial cell concentration was 1×10 6 / mL.

[0062] Immune cell activity detection method: take an appropriate amount of immune cell fluid cultured to d13, blow and disperse the agglomerate, take 100 μL, mix well with 100 μL trypan blue dye, place it on a glass slide, and examine it under a microscope (×100) Observe, take pictures, and estimate cell viability.

[0063] The cells were counted for each s...

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Abstract

The invention discloses a culture medium for culturing immune cells isolated from umbilical cord blood. The culture medium comprises a basic culture medium which is a CTS AIM-V SFM culture medium containing a serum substitute of KSR (Knockout serum replacement) with a volume concentration of 15-25% with five cytokines added therein. The invention also provides a technical scheme of mixing the culture medium with a 1640 culture medium and a culture method of immune cells isolated from umbilical cord blood. The culture medium provided by the invention has the advantages of low cost and good culture effect. The cultured immune cells have higher activity, faster growth speed and stronger killing ability. The cost of large-scale cell production is reduced.

Description

technical field [0001] The invention belongs to the field of biotechnology, and relates to a medium for culturing immune cells isolated from umbilical cord blood and a culture method thereof, in particular to an improved immune cell culture medium in which components and cytokines are mixed in an optimal ratio and its training method. Background technique [0002] Immune cells refer to cells that participate in or are related to immune responses. As an important carrier for the in vitro proliferation induction of immune cells, the culture medium is directly related to the in vitro induction results, including the number of proliferation, tumor killing effect and so on. Immune cells have the following characteristics: fast proliferation, the main utility cells CD3+CD56+T (anti-tumor activity) can proliferate in large quantities, and the cell activity is also greatly enhanced; the killing activity is strong, far superior to the traditional lymphokine activated killing Cells;...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0786
CPCC12N5/0645C12N2501/2301C12N2501/2302C12N2501/2315C12N2501/24C12N2501/515C12N2509/00
Inventor 高戎戎佟艳辉
Owner 上海中溢精准医疗科技有限公司
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