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In vitro construction method of liver organs and applications

A construction method and organoid technology, applied in cell dissociation methods, artificial cell constructs, biochemical equipment and methods, etc., to achieve the effects of increasing liver-to-body ratio, enhancing proliferation, and alleviating liver function

Active Publication Date: 2020-07-10
NANJING DRUM TOWER HOSPITAL
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  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

However, after acute liver injury such as partial hepatectomy, whether exogenous implantation of liver organoids with properties similar to hepatic progenitor cells can promote liver regeneration has not been reported yet.

Method used

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  • In vitro construction method of liver organs and applications
  • In vitro construction method of liver organs and applications
  • In vitro construction method of liver organs and applications

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0047] Organoid construction in vitro

[0048] 1. Formulation preparation

[0049] 1) Organoid induction medium: choose HepatiCuLt TM OGM Mouse Basal Medium is used as the basal medium for liver organoids, and HepatiCuLt is selected TM OGM Mouse Supplement as liver organoid basal medium supplement, choose Plasmocin TM treatment as an anti-mycoplasma reagent. HepatiCuLt TM OGM Mouse Basal Medium and HepatiCuLt TM Mix OGM Mouse Supplement according to the volume ratio of 10:1, and then add 200uL of Plasmocin TM treatment, mix well and dispense, store at -20°C.

[0050] 2) Digestive solution of liver tissue: take 45mL of Advanced DMEM / F12, add 7.5mL of Dispase and 7.5mL of Collagenase type IV and mix well.

[0051] 2. Take materials

[0052] In an ultra-clean bench, 6-8 week old C57BL / 6 normal mice were selected and killed by cervical dislocation after inhalation anesthesia. The limbs were fixed on the operating table, and the abdomen was disinfected with alcohol c...

Embodiment 2

[0068] Phenotype Characterization of Liver Organoids

[0069] 1. Gene identification:

[0070] 1) Extraction of cellular RNA

[0071] a) Remove the medium from the organoids, rinse twice with PBS to ensure that the residual medium does not affect the purity of the RNA, add 500uL Trizol to one well of the 24-well plate, and repeatedly blow and beat for full lysis, and let stand at room temperature for 10 minutes , after fully separating the protein-nucleic acid complex, transfer it to an enzyme-free 1.5mLEP tube;

[0072] b) Add chloroform at a ratio of 5:1 between Trizol and chloroform, shake vigorously up and down for 10 seconds, and let it stand at room temperature for 5 minutes to allow natural phase separation.

[0073] c) After centrifugation at 12000g for 15 minutes at 4 degrees, carefully collect the upper aqueous phase and transfer it to another new 1.5mL EP tube;

[0074] d) Add an equal volume of isopropanol to the supernatant and mix, and place at -20°C for 1 hou...

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Abstract

The invention discloses an in vitro construction method of liver organs and applications. Mouse hepatocytes are extracted to induce to construct liver organs; the forms of the liver organs are observed under a microscope; and immunofluorescence and PCR identification are performed on dryness and epithelial cell properties. An acute hepatic failure mouse model after 70% hepatectomy is established;the liver functions on the first, fourth and seventh day after operation are evaluated after the liver organs are transplanted; liver-to-body ratio is compared; and the treatment effects of the liverorgans on acute hepatic failure are verified through HE staining and immunohistochemistry. Inflammations such as HE staining and ki-67 staining and proliferation indexes show that proliferation can beobviously enhanced after the liver organs are transplanted. The liver organs have strong dryness and proliferation functions; the liver functions of acute hepatic failure mice after 70% hepatectomy can be effectively relieved, and the liver-to-body ratio can be increased; and liver regeneration and repair of acute hepatic failure after 70% hepatectomy can be promoted by relieving inflammatory infiltration of livers.

Description

technical field [0001] The invention relates to the technical field of biomedicine, in particular to an in vitro construction method and application of liver organoids. Background technique [0002] Acute liver failure (ALF) is a clinical syndrome of a large number of liver cell necrosis and severe liver damage in a short period of time caused by factors such as viruses, drugs, toxins, or alcohol, often causing jaundice, hepatic encephalopathy, and multiple organ dysfunction. Complications such as failure and high mortality. Liver transplantation is currently the only effective treatment. However, the shortage of donor livers is far from meeting clinical needs, and many patients died while waiting for liver donors. In addition, medical treatment is only symptomatic and supportive and cannot improve the prognosis of patients, so it is urgent to develop an alternative treatment. [0003] Stem cells, with their unique capabilities in self-renewal and differentiation potential...

Claims

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Application Information

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IPC IPC(8): C12N5/071A61K35/407A61P1/16
CPCC12N5/067A61K35/407A61P1/16C12N2509/00C12N5/0671C12N2500/84C12N2533/90A01K2207/30A01K2227/105A01K2267/035
Inventor 施晓雷王经琳任昊桢丁义涛
Owner NANJING DRUM TOWER HOSPITAL
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