Method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and application of method

A technique for primary hepatocytes and cell culture, used in the field of in vitro induction of primary hepatocytes bile ducts and long-term culture, expansion and differentiation

Active Publication Date: 2017-05-31
SECOND MILITARY MEDICAL UNIV OF THE PEOPLES LIBERATION ARMY
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  • 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

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  • Method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and application of method
  • Method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and application of method
  • Method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and application of method

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...

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Abstract

The invention relates to the field of bioengineering technology, and in particular to a method for in-vitro induction of cholangiocyte-like transformation of primary hepatocytes and for long-term culture, amplification and differentiation and an application of the method. The invention provides a hepatocyte cholangiocyte-like transformation medium determined by chemical ingredients and / or a system which is composed of a hepatocyte mature medium and is applicable to long-term stable culture, amplification and differentiation of the primary hepatocytes; the invention also provides the method for in-vitro induction of cholangiocyte-like transformation of the primary hepatocytes and for long-term culture, amplification and differentiation; and with the application of the method, the cholangiocyte-like hepatocyte conversion of the primary hepatocytes can be induced in vitro, so that the obtained hepatocytes have characteristics of biliary epithelial cells and hepatic precursor cells, and the hepatocytes are applicable to long-term stable culture and amplification. The breedable cholangiocyte-like hepatocytes and hepatocytes, which are mature in differentiation, prepared by the invention are applicable to such aspects as toxicologic and pharmacological evaluation of compounds and drugs, researches and diagnosis & treatment of hepatitis viruses, treatment by hepatocyte transplantation, preparation of bioartificial liver and the like.

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

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Application Information

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Patent Type & Authority Applications(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
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