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Detection method for evaluating intestine toxicity of medicine by utilizing 3D organ

An organoid, enterotoxic technology, applied in the field of biomedicine, can solve the problem of unreported safety evaluation

Inactive Publication Date: 2019-04-19
EAST CHINA NORMAL UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0008] At present, 3D organoids have been applied in many fields such as drug screening, human organ development research, disease mechanism research and personalized medicine, but there are no reports on the use of 3D organoids to study drug toxicity and safety evaluation.

Method used

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  • Detection method for evaluating intestine toxicity of medicine by utilizing 3D organ
  • Detection method for evaluating intestine toxicity of medicine by utilizing 3D organ
  • Detection method for evaluating intestine toxicity of medicine by utilizing 3D organ

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0113] Example 1 Sorting of C57BL / 6 mouse crypts and culture of 3D organoids

[0114] (1) Take the small intestine of 6-8 week-old C57BL / 6 mice, divide the small intestine into several 5-8cm long segments from the position 1-2cm away from the duodenum, remove the connective tissue on the small intestine, and Cut it in the middle, wash it with pre-cooled phosphate buffered saline (Phosphate buffered saline, PBS), then transfer it to a 50ml centrifuge tube containing an appropriate amount of PBS, and place it on ice.

[0115] (Note: All the following steps are performed in a sterile environment, and the crypts must be placed on ice as much as possible.)

[0116] (2) In a biological safety cabinet, wash the small intestine fragment with PBS containing penicillin / streptomycin (P / S) for 3-4 times, then transfer it to a 50ml centrifuge tube, add 25ml of PBS containing 2mM EDTA , digested in a refrigerator at 4°C, and after 10 minutes, transfer the small intestine fragments to a new...

Embodiment 2

[0124] Embodiment 2 Verification of 3D organoid model

[0125] 2.1 Observation of 3D organoid morphology

[0126] The crypt culture of C57BL / 6 mice was observed and photographed under the Olympus IX 71 microscope equipped with the Olympus DP 71 camera system every day from the first day to the fifth day of culture. The result is as figure 1 shown. From figure 1 Organoid structures derived from crypt differentiation can be seen in . A villous spherical cavity formed in the center of the organoid, surrounded by newly formed crypts.

[0127] 2.2 Evidence of enterotoxicity of drugs from changes in organoid morphology

[0128] (1) Preparation of drug-containing medium. A 100 mM stock solution of irinotecan was prepared in DMSO and diluted to 5 μM, 10 μM, 20 μM, 50 μM, 100 μM with complete medium containing growth factors (Responding, m-noggin, m-EGF). 10 μM stock solution of ailanthone was prepared in DMSO and diluted to 0.02 μM, 0.05 μM, 0.1 μM, 0.2 μM, 0.5 μM with complete...

Embodiment 3

[0137] Example 3 Using 3D organoids to study the enterotoxic dose of drugs

[0138] (1) Preparation of drug-containing medium. The preparation method and concentration setting are the same as "Example 2.2".

[0139] (2) On the third day of organoid culture, aspirate the original medium in the cell plate, add complete medium containing different concentrations of irinotecan or ailanthonone, set 3 parallel groups for each concentration, and set 3 blanks at the same time For medium control wells, the cell plate was placed in a 37°C cell culture incubator to continue culturing.

[0140] (3) After 48 hours of administration, the cell plate was taken out from the incubator and added to the MTS kit in the dark. Add 20 μl of MTS solution to 100 μl of culture medium in each well, shake gently after adding, and incubate for 2 hours in a 37°C cell culture incubator protected from light.

[0141] (4) After the incubation, the absorbance value of each well (the absorbance wavelength is ...

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Abstract

The invention relates to a detection method for evaluating intestine toxicity of medicine by utilizing a 3D organ model. According to the detection method, firstly, small intestinal crypt of a C57BL / 6mouse is subjected to sorting, inoculated to a culture dish containing matrigel and cultured in an Advanced DMEM / F12 culture medium, and the shape of the 3D organ is observed. The 3D organ model is verified by utilizing a positive compound, and changes of the shape of the 3D organ and IC50 detection are included. Finally, the model is utilized for conducting toxicologic study on new medicine, andthe study particularly comprises the IC50 detection of the medicine, apoptosis conditions of the 3D organ and a mechanism of medicine induction organ apoptosis. The detection method has the advantages of being simple, quick and high in sensitivity, the 3D organ mode can be combined for conducting intestine toxicity detection on the medicine, and the medicine intestine toxicity mechanism can be conducted.

Description

technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a method for evaluating intestinal toxicity of drugs by using a 3D organoid model. Background technique [0002] The intestine is the most important digestive organ in humans and the largest immune organ. The mammalian gut consists of three segments: the small intestine, large intestine, and rectum. The small intestine mainly performs a large amount of digestion, and almost all the absorption of digested products is carried out in the small intestine. The main function of the large intestine is to concentrate food residues to form feces, which are then excreted through the rectum. The intestine is not only a digestive organ, but also the largest detoxification organ in the human body. After food and drugs are taken orally, the first body barrier that comes into contact with them is the gastrointestinal tract. The gastrointestinal tract is the body's first line...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): G01N33/50C12N5/071
CPCC12N5/0679C12N2500/32C12N2500/84C12N2501/11C12N2513/00G01N33/5076
Inventor 王昕梁辰美子张远金刘明耀
Owner EAST CHINA NORMAL UNIV
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