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Peripheral blood NKT cell culture solution and culture method

An NKT cell and culture method technology, applied in the field of peripheral blood NKT cell culture medium and culture, can solve the problems of weak cell killing activity, low NKT cell content purity, low cell expansion rate, etc., and achieve strong tumor cell killing effect. , The effect of enhancing tumoricidal activity and high killing activity rate

Active Publication Date: 2021-11-26
东莞再立健生物科技有限公司
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0003] At present, NKT cell culture is to treat isolated peripheral blood mononuclear cells (PBMC) with IFN-γ for 24 hours, then add CD3 monoclonal antibody, IL-1α and IL-2 factors to stimulate and induce, and obtain a certain number NKT cells, but the final NKT cells obtained have low purity of effective cell content, weak cell killing activity, and relatively low cell expansion rate

Method used

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  • Peripheral blood NKT cell culture solution and culture method
  • Peripheral blood NKT cell culture solution and culture method
  • Peripheral blood NKT cell culture solution and culture method

Examples

Experimental program
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Effect test

Embodiment 1

[0077] On day 0, the final concentration of cytokine-containing serum-free medium CorningKBM581 lymphocytes 10% autologous serum added to the T175 cell culture flasks in a final volume of 25ml and the medium; then 5 × 10 6 Serum-free medium prepared above CorningKBM581 lymphocytes / ml, the resulting solution containing peripheral blood mononuclear cells at a concentration of 10v / v% autologous serum, left to stand placed in an incubator for 3 days, the ambient temperature of the incubator of 37 ℃, atmosphere 5% by volume of CO 2 Saturated and relative humidity of 100% CO 2 ; Wherein the cytokine comprises: a final concentration of 1000IU / ml of IL-2, a final concentration of 50ng / ml IL-15, a final concentration of 50ng / ml IL-18, a final concentration of 200ng / ml of CD3 single monoclonal antibodies, a final concentration of 2ng / ml dasatinib, the final concentration of 100IU ml POM / .

[0078] 3rd day 5, were added to cell culture flasks containing 50ml serum free medium ...

Embodiment 2

[0084] On day 0, containing a final concentration of 1% autologous serum GT-T551H3 lymphocyte serum-free medium was added to the T175 cell culture flasks in a final volume of 25ml and the medium; then 5 × 10 6 A / the above-obtained PBMC ml added to the medium, placed left to stand for 3 days in an incubator, the incubator temperature was 37 ℃, atmosphere 5% by volume of CO 2 Saturated and relative humidity of 100% CO 2 ; Wherein the cytokine comprises: a final concentration of 10IU / ml of IL-2, a final concentration of 5ng / ml of IL-15, a final concentration of 5ng / ml of IL-18, a final concentration of 5ng / ml of CD3 single monoclonal antibodies, a final concentration of 0.01ng / ml dasatinib, a final concentration of 5IU POM / ml of.

[0085] 3rd day 5, were added to cell culture flasks containing 50ml serum free medium lymphocytes and cytokines GT-T551H3, cell culture, cell concentration at 1 × 10 7 A / ml ~ 2 × 10 7 Cells / ml range; wherein the cytokine comprises: IL-2 c...

Embodiment 3

[0091] On day 0, RPMI-1640 medium containing a final concentration of 20% autologous serum was added to the T175 cell culture flask, and the final volume of the medium was 25 ml; then the 5 × 10 will then be 5 × 10 6 The above-mentioned peripheral blood mononuclear cells prepared in the above-mentioned peripheral blood cells were placed in the incubator for 3 days, and the ambient temperature of the incubator was 37 ° C, and the ambient atmosphere was a volume percent of 5% CO. 2 And relative saturation humidity is 100% CO 2 Wherein, the cytokine includes: the final concentration of 1000 qu / mL IL-15, the final concentration of 500 ng / ml of IL-18, the final concentration of 1000 ng / ml CD3 single The cloned antibody, the final concentration of 5 ng / ml, the final concentration of 500 IU / ml.

[0092] On day 3 and 5, 50 ml of RPMI-1640 medium and cytokines were added to cell culture flasks, and cell culture was carried out, and cell concentration was controlled at 1 × 10. 7 O...

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Abstract

The invention discloses a peripheral blood NKT cell culture solution and a culture method. The NKT cell culture method comprises the following steps of adding peripheral blood mononuclear cells into a lymphocyte serum-free culture medium of autoserum containing cytokines, and culturing for about 14 days; in the culture period, a culture solution containing cell factors needing to be added every 2-3 days; after culture is finished, performing centrifugal treatment and cleaning with a 0.9% sodium chloride solution to obtain peripheral blood NKT cells; adding human serum albumin into the peripheral blood NKT cells, and fixing the volume by using a 0.9% sodium chloride solution to obtain the peripheral blood NKT cells. According to the culture method, a peripheral blood single core has no immunological rejection reaction on autoserum, cells are purer, and the safety is higher; the NKT cells are fast in growth and high in amplification rate; and the NKT cell killing activity is high, and a relatively strong tumor cell killing effect is achieved.

Description

Technical field [0001] The present invention relates to the field of cytology, cell culture and in particular, relates to a method of culturing peripheral blood NKT. Background technique [0002] NKT cells (natural killer T Cell), are a class of multiple cytokines induced killer cell-mediated cytotoxic activity is most immune cells, and tumoricidal activity having a height of T lymphocytes and NK cells non-primary limiting the tumoricidal effector complex (MHC) compatible with tissue. NKT cells having proliferation speed advantages tumoricidal activity. [0003] At the moment, of NKT cells in culture is isolated peripheral blood mononuclear cells (PBMC) by IFN-γ 24 hours after treatment, CD3 monoclonal antibody was added, IL-1α and IL-2-induced stimulation factor, obtain a certain number of NKT cells, but the purity of the low content of active cells in the finally obtained NKT cells, cytotoxic activity was weak, and the relatively high rate of cell expansion. Inventive content ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0783
CPCC12N5/0646C12N2500/90C12N2501/2302C12N2501/2315C12N2501/2318C12N2501/515C12N2501/998
Inventor 王斌谢海涛谢炜豪刘元甲薛卫巍
Owner 东莞再立健生物科技有限公司
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