Isolation, Culture And Immortalization Of Sinusoidal Endothelial Cells From Mouse Liver

Objective:To establish a method for the isolation and culture of mouse liver sinusoidal endothelial cells (SEC), and study their biological characteristics.Methods:Liver tissue from mouse was treated with a 2U/mL dispase solution, and then the cell suspension was separated by Percoll density gradient centrifugation. SEC was purifed by culture in an endothelial cell selective medium and passage with enzyme discrepancy digestion. SEC immunophenotypic characterizations were analyzed using cyto-immunostaining, ultrastructures were observed under Scanning Electron Microscopy; the ability of uptaking of acetylated low-density lipoprotein (acetylated LDL) and capillary tube formation in vitro was detected.Results:The isolated cells showed the typical cobblestone morphology characteristic of endothelial cells. These cells expressed vWF, but not CD31, CD90, CD105, desmin and CK19; And uptaked acetylated low-density lipoprotein, also could form capillary-like structure. Electron microscopy showed that these cells showed typical transcellular fenestrations and W-P bodies consistent with a sinusoidal origin and endothelial cells respectively.Conclusions:We develope a method for the isolation and culture of SEC with typical morphologic and phenotypic characteristics.Objective:To study the biological characteristic of spontaneously immortalized sinusoidal endothelial cellsMethods:X-gal staining was used for evaluting senescence status of immortalized SEC. The cells were assayed for immunophenotypic characterizations by flow cytometry and cyto-immunostaining, the proliferative activity were detected by cell growth curve.The ability of uptaking of acetylated low-density lipoprotein (acetylated LDL) and formation of capillary-like structures in vitro was also detected.In order to analyze genetic stability of immortalized SEC, karyotype analysis was performed.The cells were futher cultured on soft-agar in vitro and inculated into SCID mouse in vivo to exclude tomorigenecity. Finally, P53 protein levels were detected by western blot.Results:After cultured for 8 generations in vitro, SEC became senescence.When senescent cells cultured for 40 days, small new round strong-refractive cells appeared, which showed clonal, multi-layer growth and with the increase of passage, the proliferative activity of these cells increased. All those characteristics were consistent with that of immortalized cells. The immortalized SEC expressed vWF, CD31, CD90, CD 105 and desmin, and could uptake acetylated low-density lipoprotein, but could not form capillary-like structures.Also they lacked transformed phenotypes in vitro and in vivo. Furthermore, the result of western blot showed a high level of p53 protein in immortalized SEC.Conclusions:In vitro sinusoid endothelial cells could spontaneously immortalize. Some biological characteristics of immortalized cells changed.