BACKGROUND: It is necessary to establish an effective method of isolating and culturing primary liver sinusoidal endothelial cells (LSECs) from nude mice to intensively investigate the biological features and dynamic growth state of LSECs.
OBJECTIVE: To establish a method to isolate and culture primary LSECs so as to indentify the purity and observe the biological characteristics of LSECs.  
METHODS: Liver tissues were removed from nude mice under sterile condition and cut into species of 1 mm3. Then, the species were digested with type Ⅳ collagenase to generate single cells suspension at 37 ℃. Single cells were subsequently captured by CD146 microbeads linked to LSECs with magnetic activated cells sorting. Under an inverted microscope, LSECs samples were examined to make sure the cells homogeneity and expression of von-Willebrand factor by immunocytochemistry according to LESCs’ morphology and phenotype. In addition, apoptotic LESCs were labeled with Hochest33342 under a fluorescence microscope.
RESULTS AND CONCLUSION: The captured cells from liver tissues with CD146 microbeads were positive for von-Willebrand factor and more than 95% cells were confirmed to be endothelial cells. All the cells were free from Trypan blue staining and were verified to be in good vitality. Under the inverted microscope, the LSECs showed a monophasic cobblestone feature as other endothelial patterns, and most of LSECs displayed apoptosis of Hochest33342 staining after 3 weeks. This isolation method is easy and reliable to separate LSECs from the liver of nude mice with a high purity.  

Wang HX, Deng YJ, Jiang Q, Tang N, Hu CT, Zhang JY, Ding YQ. Isolation, culture and identification of liver sinusoidal endothelial cells in nude mice. Zhongguo Zuzhi Gongcheng Yanjiu. 2012;16(15): 2789-2792.    
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