Isolation Of Stellate Cells Research Articles

Advanced method for isolation and culture of hepatic stellate cells from mice with different degrees of liver fibrosis

Objective To establish an advanced method for the isolation and culture of hepatic stellate cells (HSCs) from mice with different stages of liver fibrosis. Methods HSCs were isolatedby collagenase perfusion in combination with in vitro digestion and density gradient centrifugation. The cell purity was determined by morphological observation and HSCs’ autofluorescence. The viability of HSCs was identified by trypan-blue stain. Immunofluorescent stain was used to assess the activation state of HSCs. Results The yield, purity and viability of HSCs isolated from normal mice are 1.14×106, 95.7% and 92.8% respectively while they are 1.85×106, 90.6% and 97.3% from mice with different stages of liver fibrosis. Cells from both normal and fibrotic liver glowed well and activated gradually in vitro culture. Conclusion A simple and efficient method based on density gradient centrifugation for isolation of hepatic stellate cells from mice with different stages of liver fibrosis has been developed in this study. Key words: Mouse; Hepatic stellate cells; In situ perfusion; Density gradient centrifugation; Primary culture

A simple method for the simultaneous isolation of stellate cells and hepatocytes from rat liver tissue.

Hepatic stellate cells (HSCs), also referred to as Ito cells, perisinusiodal cells and fat-storing cells, have numerous vital functions. They are the main extracellular matrix-producing cells within the liver and are involved in the storage of retinol. HSCs are also known to secrete a number of liver mitogens. Current isolation techniques are cumbersome and most require a pronase digestion step, which destroys any hepatocytes present. We present a simple method for isolation and culture of hepatic stellate cells from the normally discarded washings from a two-step collagenase hepatocyte isolation, which has shown a yield of more than 1.5 x 10(6) viable HSCs after 5 days in culture. The cells were positively identified as HSCs by staining for two intermediate filaments (desmin and GFAP) and observing their distinct morphology from other liver cell types. This efficient method allows rapid and consistent isolation of stellate cells to give a culture that may be passaged several times.

An improved method for the purification of stellate cells from rat liver with dichloromethylene diphosphate (CL2MDP).

Hepatic perisinusoidal cell population consists of hepatic stellate cells, Kupffer cells, endothelial cells, and Pit cells. These cells are isolated by enzymic digestion and purified by density gradient centrifugation. With isolation of stellate cells, conventional method is unable to eliminate the contamination of Kupffer cells because the densities of these two cells are similar. We report here an improved method for isolation of highly purified hepatic stellate cells, using dichloromethylene diphosphate (CL2MDP), which has selective cytotoxicity of Kupffer cells. Three days after the single intravenous administration of liposome-encapsulated CL2MDP, the Kupffer cells disappeared almost completely from the liver. Following Percoll density gradient centrifugation, the purity of the hepatic stellate cells exceeded 98% without any contamination of the Kupffer cells. Kupffer cells are reported to affect the physiological functions of stellate cells. The availability of highly purified stellate cells will facilitate the investigation of their functions in primary culture.