A kind of in vitro construction method and application of liver organoids

A construction method and organoid technology, applied in cell dissociation methods, artificial cell constructs, biochemical equipment and methods, etc., to achieve the effects of enhancing proliferation, reducing inflammatory infiltration in the liver, and promoting liver regeneration and repair

Active Publication Date: 2020-12-25
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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  • A kind of in vitro construction method and application of liver organoids
  • A kind of in vitro construction method and application of liver organoids
  • A kind of in vitro construction method and application of liver organoids

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 and application of liver organoids. Mouse hepatocytes are extracted to induce the construction of liver organoids, their morphology is observed under a microscope, and their stemness and epithelial cell attributes are identified by immunofluorescence and PCR. Establish a mouse model of acute liver failure after 70% hepatectomy, evaluate the liver function on the 1st, 4th, and 7th day after liver organoid transplantation, compare the liver-to-body ratio, and verify the effect of liver organoids on acute liver failure by HE staining and immunohistochemistry. Therapeutic effects in liver failure. Inflammation and proliferation indicators such as HE staining and ki-67 staining showed that the proliferation of liver organoids was significantly enhanced after transplantation. Liver organoids have strong dryness and proliferation function; they can effectively alleviate the liver function of mice with acute liver failure after 70% hepatectomy, increase the liver-to-body ratio, and promote acute liver failure after 70% hepatectomy by reducing liver inflammation infiltration. Liver regeneration and repair in liver failure.

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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Patent Type & Authority Patents(China)
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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