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Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells

A culture method and in vitro culture technology, applied in the direction of cell culture supports/coatings, hepatocytes, artificial cell constructs, etc., can solve problems such as the reduction of enzyme function activity, and achieve the effect of improving material transfer

Inactive Publication Date: 2016-06-22
DALIAN MEDICAL UNIVERSITY
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Problems solved by technology

However, due to the reduction of enzyme functional activity, the sensitivity of hepatocyte monolayer culture method in the screening of hepatotoxic drugs is only 50%.

Method used

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  • Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells
  • Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells
  • Culturing method for improving in-vitro differentiation phenotype and function of hepatic cells

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Embodiment Construction

[0018] The specific implementation manner of the present invention will be described below with reference to the accompanying drawings. Such as Figure 1 to Figure 7 Shown:

[0019] Preparation of porous silk protein scaffold:

[0020] First prepare a silk fibroin solution with a concentration of 6-7%, and prepare a porous silk fibroin scaffold by salting out-drying method, control the pore diameter of the scaffold to 500-600 μm by the size of the salt particles, fully soak in deionized water after drying, and then A three-dimensional scaffold (ie porous silk protein scaffold) with a diameter of 5 mm and a thickness of 3 mm was prepared using a standard puncher. The porous silk protein scaffold needs to be sterilized by high temperature and high pressure before inoculating cells, and soaked in the culture medium for pre-equilibration for 12 hours.

[0021] Isolation and culture of primary rat hepatocytes:

[0022] After rats were anesthetized, two-step perfusion with colla...

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Abstract

The invention discloses a culturing method for improving the in-vitro differentiation phenotype and function of hepatic cells. The culturing method is characterized by comprising the following steps that the hepatic cells are adopted as seed cells, a porous silk protein scaffold is taken as an in-vitro culturing carrier of the hepatic cells, the hepatic cells, hepatogenic interstitial cells and an extracellular matrix are inoculated to the porous silk protein scaffold according to a certain proportion, and the porous silk protein scaffold is put in a simulated hepatic cell in-vitro culturing environment to be cultured. A three-dimensional hepatic cell co-culturing system built through the method not only can be applied to tissue-engineered hepatic cell function units, but also can be applied to in-vitro evaluation of an influence of drugs on a hepatic metabolism enzyme and hepatic toxicity detection, and therefore a novel screening and evaluating tool is supplied to new drug research and development.

Description

technical field [0001] The invention relates to an in vitro culture method of hepatocytes, in particular to a culture method capable of improving the differentiation phenotype and function of hepatocytes in vitro. Background technique [0002] Primary hepatocytes are not only an ideal seed cell source for constructing tissue engineered liver, but also an important model for accurate evaluation of ADME / T properties of drugs in vitro. However, hepatocytes are prone to "dedifferentiation" in the traditional in vitro planar culture process, that is, there is a decrease in activity, a change in phenotype, and a decrease in or even complete loss of functional activity (including synthetic and metabolic activities). Studies have confirmed that the P450 enzyme activity of freshly isolated human hepatocytes can only be maintained for 4 hours under suspension culture conditions; in the collagen-coated culture plate, the P450 enzyme expression and enzyme activity of adherent cells will...

Claims

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Application Information

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IPC IPC(8): C12N5/071C12N5/077
CPCC12N5/0671C12N2502/13C12N2533/50C12N2533/90
Inventor 王秀丽魏国峰徐红
Owner DALIAN MEDICAL UNIVERSITY
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