Animal experiment substitution method for toxicological evaluation of exogenous compounds

A technology of animal experiments and compounds, which is applied in biochemical equipment and methods, microbial measurement/inspection, microorganisms, etc., can solve the problems of long experimental period, limited detection efficiency, and human and financial resources, so as to achieve short experimental period and increase research. Value, the effect of consuming less manpower and financial resources

Inactive Publication Date: 2010-06-16
伍义行
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

Traditional toxicology evaluation methods mainly use whole animal (such as mice, rats, and dogs) experiments, which not only consume a lot of manpower and financial resources, but also have a long experiment cycle, which limits the detection efficiency and is not suitable for the research of small quantities of samples.

Method used

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  • Animal experiment substitution method for toxicological evaluation of exogenous compounds
  • Animal experiment substitution method for toxicological evaluation of exogenous compounds
  • Animal experiment substitution method for toxicological evaluation of exogenous compounds

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0011] Example 1: Isolation and culture of primary hepatocytes

[0012] 1.1 Prepare the following materials:

[0013] 1.1.1 RPMI-1640 (GIBCO TM product, Lot 2535081) culture fluid

[0014] 1.1.2 Fetal bovine serum: Hangzhou Sijiqing Bioengineering Materials Co., Ltd.

[0015] 1.1.3 Trypsin: AMRESCO product, Lot 3002B69

[0016] 1.1.4 Experimental animals: SD rats aged 1-3 days were provided by Zhejiang Experimental Animal Center.

[0017] 1.2 The preparation method is as follows:

[0018] 1.2.1 Before the experiment, after autoclaving all equipment, put the ultra-clean bench for 30 minutes of ultraviolet irradiation.

[0019] 1.2.2 After the suckling rats were soaked in 75% alcohol for disinfection, the abdomen of the suckling rats was fixed upward on the foam board, and then disinfected with iodine cotton balls, and then deiodized with alcohol cotton balls.

[0020] 1.2.3 Take out the first set of equipment (two tweezers), use two tweezers to pull the skin; then switch ...

Embodiment 2

[0026] Example 2: Compound Toxicity Detection

[0027] 2.1 Prepare the following materials:

[0028] 2.1.1 RPMI-1640 (GIBCO TM product, Lot 2535081) culture fluid

[0029] 2.1.2 Fetal bovine serum: Hangzhou Sijiqing Bioengineering Materials Co., Ltd.

[0030] 2.1.3 Test liquid (diluted with culture medium)

[0031] 2.1.4MTT (AMRESCO product, Lot 3544B04)

[0032] 2.1.5DMSO (analytical pure)

[0033] 2.2 The preparation method is as follows:

[0034] 2.2.1 Take the isolated primary hepatocytes from neonatal rats and culture them at 37°C to adhere to the wall, discard the culture medium, and add 200 μl each of different concentrations (3-6 gradients) of the test drug solution (diluted with the culture medium), 6 replicate wells were set up for each concentration, and a solvent control was set up at the same time.

[0035] 2.2.2 After continuing to culture for 48 hours: (1) Observe the cell survival with the naked eye; (2) Aspirate 100 μl of the cell supernatant as a test ...

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Abstract

The invention relates to a cell separation culture method of biological tissues and use thereof, in particular to a method for separating and culturing hepatic cells of a newborn rat (or mouse) and using the hepatic cells as a substitutional animal experiment model for toxicological evaluation of exogenous compounds. The method for separating and culturing the hepatic cells of the newborn rat (or mouse) of the invention has simple operation, does not need a liver perfusion process and expensive instruments, equipment and reagents, and can obtain more than 90% of liver parenchyma cells which have good uniformity and stability and high sensitivity and are easy for manual control. The hepatic cells separated and cultured by the invention are very sensitive to the toxicity of the exogenous compounds, have some functions and activity of in vivo hepatic cells, especially reserve the activity of drug metabolic enzyme, can well simulate the physiological environment of the in vivo liver, have the advantage of combining in vitro and integral animal toxicity tests, and can be expected to be used in high flux rapid detection of the toxicity of the exogenous compounds.

Description

1. Technical field [0001] The invention relates to a method for separating and culturing cells of biological tissues and its application, especially the method for separating and culturing neonatal rat (or mouse) hepatocytes and its method for toxicological evaluation of exogenous compounds as an alternative animal experimental model. 2. Background technology [0002] The liver is an important organ for drug metabolism under physiological and pathological conditions. After metabolism by the liver, the toxic effects of many endogenous and exogenous substances on the organism are eliminated. As an important organ for this biotransformation process, the liver must become an important target organ in toxicology research. At the same time, the liver is also the primary organ to withstand the toxic attack of exogenous compounds, and liver damage caused by exogenous toxic compounds is very common. As the main component of the liver (accounting for 90% of the weight of the liver) a...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/06C12Q1/02
Inventor 伍义行张晓梦
Owner 伍义行
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