Liver organoid model-based drug hepatotoxicity evaluation method
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An evaluation method and organoid technology, applied in the field of biomedicine, can solve the problems of limited practicability and long-term culture capacity, and achieve the effects of reducing the cost of new drug development, easy operation, and reducing labor costs.
Active Publication Date: 2020-11-24
ACCURATE INT BIOTECHNOLOGY (GUANGZHOU) CO LTD
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Hepatoma cell lines and animal models remain the "gold standard" for drug metabolism and toxicity testing, but their utility and long-term culture capabilities are limited
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Embodiment 1
[0039] This embodiment provides a method for evaluating hepatotoxicity of chloroquine phosphate based on a liver organoid model, comprising the following steps:
[0040] 1. Establishment of mouse liver organoid model:
[0041] 1) Sample cleaning: the mouse liver tissue was washed several times with normal saline containing 1% double antibody, and the obvious fat tissue was removed.
[0042] 2) Sample cutting: Cut the tissue into small pieces of about 0.5mm^3.
[0043] 3) Tissue digestion: ①Disperse the sheared tissue in normal saline, centrifuge to remove the supernatant, add collagenase, dispase and DNaseI to the precipitate, shake and digest at 37°C for 30min, add 1% green chain double antibody The normal saline was used to stop the digestion, and the cell pellet was collected; ②Continue to add collagenase, dispase and DNaseI to the pellet, shake and digest at 37°C for 2 hours, add green chain normal saline containing 1% double antibody to stop the digestion, and let it sta...
Embodiment 2
[0052] This embodiment provides a method for evaluating the hepatotoxicity of mitoxantrone based on a liver organoid model, comprising the following steps:
[0053] 1. Establishment of mouse liver organoid model:
[0054] 1) Sample cleaning: the mouse liver tissue was washed several times with normal saline containing 1% double antibody, and the obvious fat tissue was removed.
[0055] 2) Sample cutting: Cut the tissue into small pieces of about 0.5mm^3.
[0056] 3) Tissue digestion: ①Disperse the sheared tissue in normal saline, centrifuge to remove the supernatant, add collagenase, dispase and DNaseI to the precipitate, shake and digest at 37°C for 30min, add 1% green chain double antibody The normal saline was used to stop the digestion, and the cell pellet was collected; ②Continue to add collagenase, dispase and DNaseI to the pellet, shake and digest at 37°C for 2 hours, add green chain normal saline containing 1% double antibody to stop the digestion, and let it stand P...
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Abstract
The invention provides a liver organoid model-based drug hepatotoxicity evaluation method. The evaluation method comprises the steps: 1, establishing a mouse liver organoid model: (1) taking mouse liver tissue, and sequentially carrying out cleaning, shearing, digestion, lysis and filtration treatment to obtain mouse liver stem cell precipitate; (2) preparing glue from the mouse liver stem cells,and performing culturing with a culture medium to obtain a liver organoid model; and 2, carrying out drug treatment on the liver organoid model: setting a drug to be detected into a plurality of concentration gradients, then giving drug stimulation under different concentration gradients to the liver organoid model, collecting organoid samples at different time points, observing morphological changes of the organoid samples, and detecting liver function indexes. The invention provides a more accurate method for in-vitro detection of drug hepatotoxicity, which has the advantages of short periodand simple operation, can replace the traditional cell line model and animal model, and is used for preclinical new drug development and clinical drug hepatotoxicity evaluation.
Description
technical field [0001] The invention belongs to the technical field of biomedicine, and in particular relates to a drug hepatotoxicity evaluation method based on a liver organoid model. Background technique [0002] The liver's unique metabolic functions and relationship to the gastrointestinal tract make it an important target for drug and xenobiotic toxicity. Changes in liver metabolic activity from birth to adolescence with differential sensitivity to allotoxins. Hepatic drug metabolism, the often unbalanced production and detoxification process of toxic metabolites, can affect the degree of hepatotoxicity. In cases of severe toxicity, patients may develop hepatic failure. Drug-induced liver injury is currently the most common adverse event in the development of new drugs that leads to drug withdrawal due to safety, and liver toxicity discovered after approval for marketing also limits the use of many drugs. It would be a great boon to humans and experimental animals i...
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