Application of B27 additive and analogue thereof to culture of lymphocytes through serum-free media

A technology of serum-free medium and lymphocytes, which is applied in the field of cell culture, can solve the problems of poor cell culture effect and large gap in proliferation effect, and achieve large-scale culture suitable for in vitro, improve immune safety, and amplification effect better effect

Active Publication Date: 2016-03-23
无锡生基医药科技有限公司
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  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, compared with the culture conditions supplemented with serum, the proliferation effect is still quite different
[0005] Commercially available B-27 supplement (also referred to as B27) as a serum-free supplement is currently known to be used for the growth and short-term or long-term activity of hippocampa

Method used

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  • Application of B27 additive and analogue thereof to culture of lymphocytes through serum-free media
  • Application of B27 additive and analogue thereof to culture of lymphocytes through serum-free media
  • Application of B27 additive and analogue thereof to culture of lymphocytes through serum-free media

Examples

Experimental program
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Effect test

Embodiment 1

[0037] The cultivation and activation of embodiment 1PBMC

[0038] In the present invention, if there is no special instruction, the PBMC extracted from peripheral blood and modified by genetic engineering technology are activated by phytohemagglutinin (PHA) or anti-CD3 / CD28 antibody (or antibody-coupled magnetic beads). Add interleukin-2 (IL-2) (100 U / mL) to the commercially available medium of B27 (purchased from ThermoFisher, catalog number 17504044) or fetal bovine serum at 37°C and 5% CO 2 Cultured under certain conditions, the medium was changed every 2 days, interleukin-2 was supplemented, and the in vitro amplification was performed for 10-20 days.

Embodiment 2B27

[0039] Example 2B27 shows similar or better effects on PBMC proliferation

[0040] In 4 different commercially available media AIM-V (product number 12055-091, medium A), X-VIVO (product number 04-418Q, medium B), TexMACS (product number 170- 076-309, medium C) and GT-T551H3 (Product No. WK593S, medium D), were added with 10% fetal bovine serum or 4% B27, respectively, and cultured primary PBMCs for 15 days as described in Example 1. Cumulative cell expansion fold was measured by counting total cells.

[0041] Such as figure 1 As shown, when PBMCs were cultured with the commercial medium without any addition, it was basically difficult to maintain the proliferation of PBMCs. When culturing PBMCs in medium A or B, B27 and fetal bovine serum showed similar effects on PBMC proliferation, such as lymphocyte proliferation over 200 times within 7 days and over 400 times within 14 days; When culturing PBMCs in C and D, fetal bovine serum cannot maintain the proliferation of PBMCs,...

Embodiment 4B27

[0055] Example 4B27 maintains a dose-dependent relationship in the proliferation of PBMCs

[0056] PBMC were cultured in X-VIVO15 (product number 04-418Q, medium B) and TexMACS (product number 170-076-309, medium C) according to the method described in Example 1, with different concentrations of B27 PBMCs were used for 12 days, and the cumulative cell expansion times were measured every 3 days. Cumulative cell expansion fold was measured by counting total cells.

[0057] Such as image 3 As shown, higher concentrations of B27 (such as 2% and 4%) in medium B caused more than 1600-fold proliferation of PBMCs, while low concentrations of B27 (such as 0.25%, 0.5%, 1%, 1.25%, 1.5 % and 1.75%) are also sufficient to maintain PBMC proliferation (>800-fold expansion). Similarly, higher concentrations of B27 (such as 2% and 4%) in medium C caused more than 650-fold proliferation of PBMCs, while low concentrations of B27 (such as 0.25%, 0.5%, 1%, 1.25%, 1.5 % and 1.75%) are also suf...

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Abstract

The invention provides application of a B27 additive and an analogue thereof to culture of lymphocytes through serum-free media. The application solves the problem that through existing lymphocyte serum-free media, expansion in vitro of lymphocytes is slow without any serum replacing additives, and the number of cells required for a clinical cell therapy is hard to meet.

Description

technical field [0001] The invention relates to the field of cell culture, and more specifically relates to the application of B27 additive and its analogs in cultivating lymphocytes in serum-free medium. Background technique [0002] In recent years, with the rapid development of immunotherapy technology, various cellular immunotherapy based on lymphocytes has become the focus of increasing attention. Immunotherapy technology based on lymphocyte technology and various lymphocytes modified by genetic engineering technology has become a powerful and effective means of anti-tumor treatment, and has shown outstanding curative effect in clinical treatment trials for refractory and relapsed leukemia patients. However, the effect of lymphocyte immunotherapy depends on a sufficient number of lymphocytes, which makes the effective expansion of lymphocytes in vitro the key technology of all cellular immunotherapy. [0003] Lymphocytes extracted from the body need to be activated in ...

Claims

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Application Information

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IPC IPC(8): C12N5/078
CPCC12N5/0634C12N2500/05C12N2500/24C12N2500/30C12N2500/34C12N2500/36C12N2500/38C12N2500/90C12N2501/71
Inventor 姚树元高昱晟林志财
Owner 无锡生基医药科技有限公司
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