Unlock instant, AI-driven research and patent intelligence for your innovation.

Method for inducing primary hepatocyte cholangiosis in vitro and long-term culture, expansion and differentiation and its application

A technology for primary hepatocytes and cell culture, applied in animal cells, vertebrate cells, artificial cell constructs, etc.

Active Publication Date: 2019-08-30
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
View PDF4 Cites 0 Cited by
  • Summary
  • Abstract
  • Description
  • Claims
  • Application Information

AI Technical Summary

Problems solved by technology

[0005] At present, there is no literature report on the method of inducing primary hepatocyte cholangiosis in vitro, and long-term culture, expansion and differentiation

Method used

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
View more

Image

Smart Image Click on the blue labels to locate them in the text.
Viewing Examples
Smart Image
  • Method for inducing primary hepatocyte cholangiosis in vitro and long-term culture, expansion and differentiation and its application
  • Method for inducing primary hepatocyte cholangiosis in vitro and long-term culture, expansion and differentiation and its application
  • Method for inducing primary hepatocyte cholangiosis in vitro and long-term culture, expansion and differentiation and its application

Examples

Experimental program
Comparison scheme
Effect test

Embodiment 1

[0138] Example 1: Culture of mouse primary hepatocytes

[0139] Use 1.25% Matrigel or 20 μg / ml Laminin to coat the culture dish, preferably to cover the bottom surface, and place it at 37° C. for one hour before use.

[0140] Mouse primary hepatocytes were isolated using a two-step collagenase perfusion method.

[0141] Inoculate 1-2×10 4 / cm 2 Hepatic parenchymal cells were transferred to the aforementioned coated culture dish, and the hepatocyte cholangiogenic medium was DMEM / F12 (Invitrogen Company), containing 1% insulin-transferrin-sodium selenite mixed solution (Invitrogen Company), 50ng / ml Epidermal growth factor (Peprotech company), 20ng / ml hepatocyte growth factor (Peprotech company), 20ng / ml fibroblast growth factor-2 (Peprotech company), 20ng / ml interleukin-6 (Peprotech company), 20ng / ml inhibitory Oncocin M (Peprotech Company), 200ng / ml Shh protein (Peprotech Company), 1 μM SAG (MCE Company) or 1 μM purmorphamine (MCE Company), 5 μg / ml Jagged-1 (Abcam Company) o...

Embodiment 2

[0150] Example 2: Inducing the mouse bile duct-like hepatocytes obtained in Example 1 to differentiate into mature hepatocytes

[0151] When the confluence rate of the mouse bile duct-like hepatocytes cultured in Example 1 reaches 90-100%, the medium is replaced with a hepatocyte maturation medium, and the medium is DMEM / F12 (Invitrogen Company), containing 1% insulin-transferrin -Sodium selenite mixed solution (Invitrogen Company), 20ng / ml Oncostatin M (Peprotech Company), 10 μ M of SB431542 (Secllek Company) or 1 μ M of A83-01 (MCE Company) or 2 μ M of RepSox (MCE Company), 1 μM LY-411575 (MCE Company), 10 μM DAPT (Selleck Company) or 1 μM Compound E (Enzo Company), 10 μM dexamethasone (Sigma Company) or 1 μM hydrocortisone (Sigma Company), 10) mA of propane Valeric acid or 1 or vorinostat acid or 1 linotrichostatin. Change the medium every two days.

[0152] After 2-3 weeks of differentiation culture, RT-PCR detected the expression levels of liver cell-related genes at di...

Embodiment 3

[0156] Example 3: Expansion of primary human hepatocytes

[0157] Petri dishes were coated as described in Example 1.

[0158] Isolation of human hepatocytes: fresh primary human hepatocytes were isolated by two-step perfusion with collagenase (Maurel P., Hepatocytes-Methods and Protocols, METHODS IN MOLECULAR BIOLOGY, ISSN 1064-3745).

[0159] The specific method is as follows: firstly, under the pressure provided by the peristaltic pump, the fresh liver tissue was rinsed continuously for 10 minutes by using the exposed lumen of the liver surface, and then the PBS was replaced with Hanks solution without calcium and magnesium ions to perfuse the liver tissue for 10 minutes, and then Add 1% BSA by mass volume ratio and 0.1% type IV collagenase (Sigma Company, USA) by mass volume ratio to perfuse for 30 minutes. The hepatocytes were gently separated from the liver tissue with a glue stick, centrifuged at 500g for 1 minute and repeated 3 times, and the precipitated cells were t...

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

PUM

No PUM Login to View More

Abstract

A method for in-vitro induction of ductal metaplasia of a primary hepatocyte and for long-term cultivation, proliferation and differentiation thereof, and media used for hepatocyte ductal metaplasia and hepatocyte maturation are provided. A ductular hepatocyte and a differentiated mature hepatocyte prepared by the above method are also provided.

Description

technical field [0001] The invention relates to the technical field of bioengineering, in particular to a method for inducing primary hepatocyte cholangiosis in vitro and long-term culture, expansion and differentiation and its application. Background technique [0002] The liver has a strong regenerative capacity. Under normal circumstances, liver homeostasis is maintained and after hepatectomy, liver regeneration is mainly completed by mature hepatocytes. In chronic liver injury, liver regeneration manifests as bile duct-like hyperplasia. Bile duct-like cells are derived from bile duct and hepatocytes, and hepatocyte-derived cholangioid hepatocytes have been shown to play a major role in liver regeneration (Tarlow, B.D. et al. Bipotential adult liver progenitors are derived from chronically injured mature hepatocytes. Cell stem cell 15, 605-618, 2014). The above shows that liver regeneration is mainly completed by hepatocytes, and the regeneration takes place in two forms...

Claims

the structure of the environmentally friendly knitted fabric provided by the present invention; figure 2 Flow chart of the yarn wrapping machine for environmentally friendly knitted fabrics and storage devices; image 3 Is the parameter map of the yarn covering machine
Login to View More

Application Information

Patent Timeline
no application Login to View More
Patent Type & Authority Patents(China)
IPC IPC(8): C12N5/071G01N33/68A61L27/38
Inventor 鄢和新王红阳
Owner SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY