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Human liver microsome and cell co-culture system and construction method and application thereof

A technology of co-culture system and human liver microsomes, applied in the field of co-culture system of human liver microsomes and cells and its construction

Pending Publication Date: 2021-11-09
ZHENGZHOU TOBACCO RES INST OF CNTC
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Currently, there are few reports on the use of human liver microsomes in sterile cell experiments

Method used

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  • Human liver microsome and cell co-culture system and construction method and application thereof
  • Human liver microsome and cell co-culture system and construction method and application thereof
  • Human liver microsome and cell co-culture system and construction method and application thereof

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0029] Example 1 Construction method of human liver microsome and Beas-2b cell co-culture system

[0030] This embodiment provides a method for constructing a co-culture system of human liver microsomes and cells, comprising the following steps:

[0031] (1) Prepare 250 μg / mL minocycline hydrochloride aqueous solution. Commercially available minocycline hydrochloride was dissolved in sterile water to prepare a 250 μg / mL minocycline hydrochloride aqueous solution, which was placed in a sterile centrifuge tube.

[0032] Prepare 10kU / mL penicillin aqueous solution. Dissolve commercially available penicillin in sterile water, prepare 10kU / mL penicillin aqueous solution, and put it in a sterile centrifuge tube.

[0033] Prepare 10mg / mL streptomycin aqueous solution. Dissolve commercially available streptomycin in sterile water, prepare 10 mg / mL penicillin aqueous solution, and put it in a sterile centrifuge tube.

[0034] Prepare NADPH regeneration system A+B mixture. Mix liqu...

Embodiment 2

[0040] Example 2 Application of Exogenous Substances in Vitro Metabolism

[0041] This example provides the application of the construction method of the human liver microsome and cell co-culture system in the in vitro metabolism of the tested exogenous substance, including the following steps:

[0042] Step 1) see embodiment 1;

[0043]2) Add the test exogenous substance to the incubation metabolism medium solution containing human liver microsomes, the incubation metabolism medium solution consists of the test exogenous substance and basal medium, minocycline hydrochloride, penicillin, Composed of streptomycin, NADPH regeneration system A+B mixture, and human liver microsomes, wherein the exogenous substance to be tested is nicotine, the final concentration of nicotine is 8 μg / mL, and the final concentration of minocycline hydrochloride The final concentration of penicillin is 100U / mL, the final concentration of streptomycin is 100μg / mL, the volume percent concentration of ...

Embodiment 3

[0047] Example 3 Human liver microsome and cell co-culture system

[0048] This embodiment provides a co-culture system of human liver microsomes and cells, which consists of basal medium, minocycline hydrochloride, penicillin, streptomycin, NADPH regeneration system A+B mixture, human liver microsomes and Beas -2b cell composition, wherein the final concentration of minocycline hydrochloride is 2.5 μg / mL, the final concentration of penicillin is 100 U / mL, the final concentration of streptomycin is 100 μg / mL, and NADPH regeneration system A+B is mixed The volume percent concentration of the solution was 6%, and the final concentration of human liver microsomes was 1 mg / mL.

[0049] In other embodiments, the co-culture system also includes a test exogenous substance; the test foreign substance is nicotine, and the final concentration of nicotine is 8 μg / mL; in another embodiment, the test foreign substance is tobacco The total particulate matter in product smoke, the final con...

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Abstract

The invention relates to a human liver microsome and cell co-culture system and a construction method and application thereof. Sterility of a human liver source is very difficult to ensure, and when human-derived liver microsome is prepared, the sterile liver microsome is hardly obtained. In order to carry out in-vitro metabolism test research on human-derived liver microsome and cell co-culture, the invention provides a co-culture method capable of enabling the germy human liver microsome to be in direct contact with sterile cells, and successfully constructs the human liver microsome and cell co-culture system. Tests prove that the construction method disclosed by the invention can ensure that pollution sources in the human liver microsome cannot influence the sterile environment of the cells, and the sterile state of normal growth of the cells is maintained. Moreover, the method is suitable for in-vitro metabolism research of allogenic substances (such as nicotine and smoke), and lays a foundation for in-vitro metabolism research of the combined action of the human-derived liver microsome and the cells.

Description

technical field [0001] The invention belongs to the technical field of cell co-cultivation, and in particular relates to a co-cultivation system of human liver microsomes and cells and its construction method and application. Background technique [0002] Liver microsomes are small vesicles obtained from fragments of the endoplasmic reticulum of liver cells, with a diameter of about 0.1 μm, and have a complete set of phase I metabolic enzymes (such as cytochrome P450, flavin monooxygenase, monoamine oxidase, etc.), phase II metabolic enzymes (such as glucuronosyltransferase, sulfotransferase), esterase, etc. These enzymes participate in drug metabolism in the human body, and are research tools designated by the FDA to predict the metabolism and inhibition tests in the human body. [0003] When conducting metabolic activation experiments involving cells, the sterile mouse liver microsomes + metabolic activation system and foreign substances are usually added to the medium to ...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071
CPCC12N5/067C12N2502/27
Inventor 华辰凤赵俊伟史清照尚平平李翔谢复炜秦亚琼王昇刘惠民
Owner ZHENGZHOU TOBACCO RES INST OF CNTC
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