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Culture method of intestinal organoids and application of intestinal organoids in intestinal toxicity test

A culture method and organoid technology, which is applied in the field of biosafety detection and evaluation, can solve problems such as the difference in the formation rate of intestinal organoids, and achieve the effects of sensitive toxicity response, expanded application range, and improved repeatability

Pending Publication Date: 2022-04-12
SUN YAT SEN UNIV
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

refer to figure 1 A It can be found that the formation rate of intestinal organoids in different studies is significantly different and generally low

Method used

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  • Culture method of intestinal organoids and application of intestinal organoids in intestinal toxicity test
  • Culture method of intestinal organoids and application of intestinal organoids in intestinal toxicity test
  • Culture method of intestinal organoids and application of intestinal organoids in intestinal toxicity test

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0037] Example 1 Culture method of mouse intestinal organoids

[0038] This embodiment provides a method for culturing mouse intestinal organoids, comprising the following steps:

[0039] 1) Separation of intestinal crypts in mice: after 6-10 weeks of mice were sacrificed, a jejunum section with a length of 10 cm was taken from the end near the stomach, and after the mesentery and fat were removed, the intestinal tissue was dissected longitudinally, rinsed and scraped off For intestinal villi, cut the intestinal tissue into 2-5mm long tissue fragments and transfer them to a centrifuge tube, wash them with cold PBS solution, add cold PBS solution containing 2mM EDTA to dissociate the tissue, and discard the supernatant after incubation on a shaker; Add cold PBS solution containing 0.1% BSA, shake vigorously to fully release the crypts from the tissue, take the supernatant and filter it through a filter with a pore size of 70 μm, collect the filtrate, and add cold PBS solution c...

Embodiment 2

[0042] Example 2 The method of using intestinal organoids to test the intestinal toxicity of heavy metals

[0043] This example provides a method for testing the intestinal toxicity of heavy metals using intestinal organoids, including the following steps:

[0044] 1) Intestinal organoids were obtained by culturing as described in Example 1;

[0045] 2) performing toxicity tests on the intestinal organoids treated with substances containing different concentrations of heavy metals, and measuring the change curve of the intestinal organoid model toxicity damage index with the concentration of heavy metals;

[0046] 3) Based on the dose-response relationship data obtained from the change curve, a Markov chain Monte Carlo simulation algorithm is used to establish a dose-response relationship curve, and the baseline dose corresponding to a 10% change in toxic effects is estimated.

[0047] In step 2) of this embodiment, the toxic damage of the cell model includes: clone formation...

Embodiment 3

[0055] Example 3 Application of Intestinal Organoids to Evaluate the Enterotoxicity of Hexavalent Chromium at Environmental Exposure Levels

[0056] Hexavalent chromium (Cr-VI) is a typical environmental pollutant, and non-occupational people are often exposed to Cr-VI through ingestion and drinking. Acute exposure to large doses of Cr-VI will cause adverse health effects of the gastrointestinal tract such as irritation, diarrhea, and acute bleeding. Long-term low-dose exposure to Cr-VI will significantly increase the risk of gastrointestinal tumors. Cr-VI has been listed as one of the priority water pollutants by many countries. At present, as the current health standard in China, the maximum allowable pollution level of Cr-VI in drinking water is 50 μg / L. However, due to fluctuations in seasonal precipitation and industrial production, the concentration of hexavalent chromium in drinking water in many regions often exceeds the standard. Therefore, the environmental polluti...

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Abstract

The invention provides a culture method of intestinal organs. The culture method comprises the following steps: 1) mouse intestinal crypt separation; and 2) culturing the intestinal organs. The invention belongs to the technical field of biological safety detection and evaluation, and provides a culture method of intestinal organs capable of overcoming subjective deviation and improving the formation rate of the organs by carrying out clear quality control and condition optimization on fraction and inoculation density determination and the like. The invention further provides application of the intestinal organ in chemical intestinal toxicity testing and a corresponding testing method.

Description

technical field [0001] The invention belongs to the technical field of biosafety detection and evaluation, and in particular relates to a method for culturing intestinal organoids and its application in intestinal toxicity testing. Background technique [0002] Organoids are in vitro 3D cultures derived from stem cells, by isolating stem cells (including: adult stem cells, embryonic stem cells, induced pluripotent stem cells) from human or mouse tissues, and relying on matrix support such as hydrogel in an in vitro culture system , Artificially adding a variety of cytokines to promote cell proliferation and differentiation and then spontaneous formation. As a new research model, organoids can simulate the structure and function of natural organs, and reproduce some key cell types and tissue structure characteristics of the represented organs, which can make up for the shortcomings of mouse models. [0003] At present, organoids derived from lung, stomach, liver, pancreas, i...

Claims

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Application Information

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Patent Type & Authority Applications(China)
IPC IPC(8): C12N5/071C12Q1/02
Inventor 陈雯王紫微陈燊张睿陈丽萍李道传蒋欣航
Owner SUN YAT SEN UNIV
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