Cultivation system for in-vitro amplification of lymphocytes and amplification method and application

A culture system and lymphocyte technology, applied in cell culture active agents, cells modified by introducing foreign genetic material, animal cells, etc., can solve the problem of difficulty in obtaining a sufficient number of NK cells, and achieve uniform quality, strong lethality, high purity effect

Active Publication Date: 2017-09-19
杭州中赢生物医疗科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

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Problems solved by technology

[0004] One of the main obstacles restricting the clinical application of

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  • Cultivation system for in-vitro amplification of lymphocytes and amplification method and application
  • Cultivation system for in-vitro amplification of lymphocytes and amplification method and application
  • Cultivation system for in-vitro amplification of lymphocytes and amplification method and application

Examples

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Example Embodiment

[0056] Example 1 Preparation of genetically engineered cells

[0057] The preparation method of genetically engineered cells is as follows: First, construct a vector that can stably express transmembrane interleukin 21, interleukin IL12A, transmembrane interleukin 12B and CD80. There is a selection marker gene on the vector. Transmembrane interleukin 21 or transmembrane interleukin 12B is connected to the cell membrane through the transmembrane portion of CD4, and interleukins IL12A and CD80 are transmembrane proteins. These vectors were transfected into K562 cells, and high-expressing cell clones were selected with antibiotics and flow cytometry.

Example Embodiment

[0058] Example 2 Preparation of lymphocyte expansion culture system

[0059] Using the genetically engineered cells prepared in Example 1 to amplify lymphocytes includes the following steps:

[0060] (1) Inactivated genetically engineered cells: irradiated with 100Gy radiation for 30 minutes to obtain inactivated K562 engineered cells;

[0061] (2) Preparation of the culture medium: take the lymphocyte culture medium RPMI1640 and 10% fetal bovine serum, and then add the inactivated K562 engineered cells in step (1) to the mixed medium, the addition amount is 1×10 6 / mL, then add IL-2, the amount of IL-2 added is 50U / mL.

Example Embodiment

[0062] Example 3 Amplification of Lymphocytes in Healthy Volunteers

[0063] (1) Cultivation: Collect fresh blood from healthy volunteers, collect serum by centrifugation and store, and obtain human peripheral blood mononuclear cells (PBMC) through lymphatic separation fluid. The inoculation density is 1×10 6 / mL (according to the ratio of the K562 engineered cells to the monocytes is 1:1), inoculated into the culture system of expanded lymphocytes in vitro;

[0064] (2) Amplification: 37°C, 5% CO 2 Cultivate for 7 days under the conditions, then add the above-mentioned culture system, and cultivate for another 7 days;

[0065] (3) Harvest: After culturing, NK cells are collected by centrifugation.

[0066] NK cells co-cultured with PBMC cells and K562 cells transfected with transmembrane interleukin 21 and CD137L were used as controls;

[0067] Use an automatic cell counter to count the expanded NK cells and draw a curve. The growth curve is like figure 2 As shown, it can be seen that...

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Abstract

The invention relates to the field of cellular immunotherapy, in particular to a cultivation system for in-vitro amplification of lymphocytes, an amplification method and application. The cultivation system comprises a mixture of interleukin 21, interleukin 12 and CD80. NK cells amplified through the cultivation system are high in purity and high in killing ability for tumor cells; on the 14th day, the NK cells can be amplified by about 9,100 times, so as to meet various application requirements of expected clinical treating effects on diseases; the problem on safety of the cultivation system for clinical application is solved, and the application cost of the NK cells is reduced; a foundation is laid for broad spectrum application of the NK cells to adoptive immunotherapy.

Description

technical field [0001] The invention relates to the field of cellular immunotherapy, in particular to a culture system for expanding lymphocytes in vitro, an expansion method and application. Background technique [0002] NK cells (natural killer cells, Natural Killer Cell) are also called large granular lymphocytes. And γδT cells, NKT cells act as a bridge between acquired immunity and innate immunity in form and function. On the one hand, when the body is infected or traumatized, NK cells, as defenders, quickly, extensively and specifically recognize antigens through non-peptide-MHC recognition methods, clear pathogenic microorganisms and mutant cells in a timely manner, and play the role of innate immunity. On the other hand, NK cells are considered to be partly involved in the adaptive immune response and can affect the effector functions of αβT cells and B cells. [0003] During the occurrence and development of tumors, NK cells can either directly recognize tumor cel...

Claims

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Application Information

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IPC IPC(8): C12N5/0783C12N5/10A61K35/17A61P35/00A61P35/02A61P31/04A61P31/12A61P31/10A61P33/00A61P1/16A61P31/20A61P31/18A61P31/14
CPCA61K35/17C12N5/0646C12N2501/2302C12N2501/2312C12N2501/2321C12N2501/51C12N2502/99
Inventor 王冶陶吴忠福彭昉刘慧显王俊俊俞英豪
Owner 杭州中赢生物医疗科技有限公司
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