Co-culturing method of human primary hepatocytes and liver nonparenchymal cells

Abstract

The invention discloses a co-culturing method of human primary hepatocytes and liver nonparenchymal cells. The co-culturing method comprises following steps: fresh hepatic tissue is washed thoroughly with exposed blood vessel perfusate I and perfusate II until elasticity of the hepatic tissue is lost, and complete digestion is realized; the hepatic tissue is delivered into a culture dish filled with a cell washing liquor, and is screened so as to obtain a single-cell suspension; a culture plate or a culture dish coated with collagen is inoculated with the single-cell suspension with a low inoculum density so as to obtain monolayer co-cultured cells with a fusion degree of 100% in the presence of growth factors; and a maintaining culture medium containing 2% DMSO is added into the culture plate or the culture dish for culturing, and then a liver island structure is formed by accumulation of hepatic cells, wherein the liver island structure is surrounded and invaded by liver nonparenchymal cells. Routine hepatocyte separation technology is employed in the co-culturing method, in vitro long-term culturing of hepatocytes as long as 120 days are realized, and HBV susceptibility is maintained for as long as 72 days. In addition, the co-culturing method can be used for screening and evaluating anti-HBV medicines, and possesses significant importance on researches of antiviral medicines.

Description

technical field [0001] The invention relates to a cell culture method, in particular to a simple co-cultivation method of hepatocytes and liver non-parenchymal cells that can maintain the characteristics of human primary hepatocytes and the susceptibility to HBV for a long time. Background technique [0002] Liver cells can be divided into hepatic parenchymal cells and hepatic non-parenchymal cells in terms of function. Cells, sinusoidal endothelial cells, hepatic epithelial cells, Kupffer cells, etc., mainly perform the functions of auxiliary liver cells, liver immunity and liver structural support. So far, the traditional co-culture method is to mix and culture pure primary hepatocytes and pure hepatic non-parenchymal cells in a certain proportion. Therefore, the morphological characteristics and physiological functions of primary hepatocytes can be maximally maintained. However, this traditional co-culture system has serious disadvantages, such as limited cell lifespan,...

AI Technical Summary

Problems solved by technology

Due to the serious dedifferentiation of these cell lines, compared with the liver cells in vivo, the differentiation state is far different, so the real antiviral effect and cytotoxicity of the drug cannot be reflected in the screening and evaluation of anti-HBV drugs

To make matters worse, these cell lines do not support HBV infection and cannot be used for research on anti-HBV infectious drugs such as small peptides, antibodies, etc.

Human primary hepatocytes are the most ideal cell infection model for HBV research. A large number of studies have shown that primary hepatocytes can be infected by HBV, but they quickly lose their differentiation characteristics when cultured in vitro, and the susceptibility to HBV is usually within 1 week. It is completely lost, which seriously restricts the establishment of cell models for the study of HBV infection mechanism, drug screening and evaluation

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