Method for in vitro evaluation of substance safety using human immortalized myeloid cells

An immortalization and material technology, applied in biochemical equipment and methods, microbial measurement/testing, biological testing, etc., can solve problems such as the absence of immune cells, difficulty in stable supply, individual differences, etc., and achieve precision and high sensitivity Evaluate and reduce the effect of error

Pending Publication Date: 2022-07-08
迈凯恩技术有限公司 +1
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0020] In addition, immune cells function through cytokines released, and currently there is no system that can test the direct effects of drugs and foods on immune cells
[0021] For example, in order to evaluate the effect of food materials on the function of immune cells, there is a method using human peripheral blood, but because blood obtained through blood donation is used, it is difficult to supply it stably, and individual differences may occur

Method used

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  • Method for in vitro evaluation of substance safety using human immortalized myeloid cells
  • Method for in vitro evaluation of substance safety using human immortalized myeloid cells
  • Method for in vitro evaluation of substance safety using human immortalized myeloid cells

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0139] (Example 1) Preparation of immortalized monocytes derived from human peripheral blood and preparation of dendritic cells

[0140] (Preparation of Immortalized Monocytes from Human Peripheral Blood)

[0141] Immortalized monocytes derived from human peripheral blood were prepared with reference to existing reports (WO2012 / 043651 and Japanese Patent Laid-Open No. 2017-131136). Specifically, CD14-positive fractions were extracted from human peripheral blood, and the genes (c-MYC, BMI1, and BCL-2) reported for the production of human immortalized monocytes were introduced into them according to existing reports, thereby producing immortalized cells. monocytes. Immortalized monocyte cell lines were grown in cells containing 20% ​​FBS (Cytiva Inc., Cat#: SH30088.03), 50 ng / mL M-CSF (Peprotech Inc., Cat#: AF-300-25), and 50 ng / mL GM - Culture in α-MEM medium of CSF (Peprotech Inc., Cat#: 300-03), obtain proliferative cells 3 to 5 weeks after the start of culture, and use a c...

Embodiment 2

[0146] (Example 2) (Preparation of immortalized monocytes derived from human induced pluripotent stem cells)

[0147] Immortalized monocytes derived from human induced pluripotent stem (iPS) cells were prepared with reference to existing reports (WO2012 / 043651, JP 2017-131136 and JP 2018-171005). Specifically, undifferentiated iPS cells (provided by the iPS Cell Research Institute of Kyoto University) were seeded in 10 cm diameter petri dishes (AGC TECHNO GLASS CO., LTD., Cat#: 1012-100) or 6-well plates (Corning Inc., Cat#: 3516), culture dishes or 6-well plates were pre-coated with laminin 511 (iMatrix-511-E8, Nippi, Cat#: 892-012) as a basement membrane, and cultured in α-MEM. 20% FBS (Cytiva Inc., Cat#: SH30088.03) was added to the base (Fujifilm Wako Pure Chemical Industries, Ltd., Cat#: 137-17215) to obtain a culture medium (hereinafter referred to as α-MEM medium (containing 20 %FBS)), use this medium to start differentiation induction culture. After that, the α-MEM m...

Embodiment 3

[0150] (Example 3) Production of immortalized monocytes derived from human induced pluripotent stem cells expressing M-CSF and GM-CSF

[0151] Immortalized monocytes derived from human induced pluripotent stem cells expressing M-CSF and GM-CSF were prepared with reference to the existing report (Japanese Patent Laid-Open No. 2018-171005). Specifically, using a third-generation lentiviral vector (SignaGen Inc.) in which the promoter for expressing the protein is EF1a, the human induced pluripotent stem cell-derived immortalized monocytes obtained in Example 2 were simultaneously introduced with human M. - Expression vector of CSF gene and human GM-CSF gene, wherein the third-generation lentiviral vector is a strain of human immunodeficiency virus type 1 (HIV-I) lacking in proliferation ability and the like. 3 days after gene introduction, the cells were cultured in α-MEM (containing 20% ​​FBS) medium that did not contain human M-CSF and GM-CSF, and 3 to 5 weeks after the start ...

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Abstract

The invention relates to a method for evaluating the safety of substances in vitro, and discloses a method which is more stable, reproducible, economical and easy to operate by using human immortalized myeloid cells. The invention relates to a method for evaluating skin sensitization and/or fever of a substance to be detected by using human immortalized myeloid cells, a method for detecting skin sensitization substances and/or fever substances in a sample, and a method for evaluating the effect of the sample on the function of immune cells. The method comprises the step of measuring the yield of IL-6 and/or IL-8 in a culture solution of human immortalized myeloid cells.

Description

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS [0002] This international application claims priority based on Japanese Patent Application No. 2019-208711 filed with the Japan Patent Office on November 19, 2019, the entire contents of which are incorporated herein by reference. technical field [0003] The present invention relates to a method for evaluating the skin sensitization and / or exothermicity of a substance in vitro using human immortalized myeloid cells, a method for detecting a skin sensitizing substance and / or exothermic substance in a sample, and a method for evaluating the effect of a sample on the Methods for the effects of immune cell function. Background technique [0004] When providing products and substances for human consumption, etc. to eat or apply, it is necessary to confirm in advance whether the substances contained in the products are safe and whether they will cause allergic reactions, etc. [0005] In the test method, for example, as a method f...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/0784
CPCG01N33/6869G01N33/5047G01N2333/5412G01N2333/5421G01N33/6863C12N2510/00C12N2501/22C12N2501/2304C12N2533/52C12N2506/45C12N5/0647C12N2510/04G01N33/5038G01N33/54386
Inventor 宫崎和雄清水淳田中善孝
Owner 迈凯恩技术有限公司
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