Culture and characterization of sinusoidal endothelial cells isolated from human liver

Abstract

Although most vascular models use large vessel endothelial cells from human umbilical veins, there is marked heterogeneity among endothelial cells from different vascular beds and organs. More accurate modeling of endothelial involvement in liver diseases, including metastasis, may result from the use of human hepatic sinusoidal endothelial cells. Liver resection specimens were sectioned, then treated with a 1.2 U/ml dispase solution. The tissue slurry was mechanically disaggregated and separated by centrifugation on a Percoll density gradient. Cells were then cultured in an endothelial-specific media with growth factors. These techniques resulted in a homogeneous monolayer consistent with endothelial cells by light microscopy. An endothelial origin was further confirmed by the expression of Factor VIII, binding of Ulex lectin, and uptake of acetylated low density lipoprotein. Electron microscopy showed transcellular fenestrations consistent with a sinusoidal origin. These human hepatic sinusoidal endothelial cells were then studied for expression of the adhesion molecules CD31/PECAM, CD34, E-selectin, ICAM-1, L-selectin, LFA-3, P-selectin, and VCAM-1 plus the binding of wheat germ agglutinin lectin. The patterns of adhesion molecule expression and lectin binding by these cells are characteristic of hepatic sinusoidal endothelia. In this paper, we have described a method for isolation and culture of human cells with the morphologic and phenotypic characteristics of hepatic sinusoidal endothelia.

Immunohistochemical Heterogeneity of the Endothelium of Blood and Lymphatic Vessels in the Developing Human Liver and in Adulthood

“…From the 10th week of gestation, the expression of CD31 and CD34 is present only on the portal blood vessels, but almost nonexistent in the endothelial cells of the liver sinusoids. The absence of CD34 immunopositivity on the endothelial cells of hepatic sinusoids has been confirmed in studies performed by other researchers [Couvelard et al, 1993;Daneker et al, 1998]. Pusztaszeri et al [2006] demonstrated CD31 immunoreactivity on sinusoidal endothelial cells in healthy adults, whereas CD34 immunoreactivity is present only in the endothelial cells of periportal sinusoids.…”

Honokiol, a Small Molecular Weight Natural Product, Inhibits Angiogenesis in Vitro and Tumor Growth in Vivo

“…TRAIL Inhibition Studies (Endothelial Proliferation, VEGFR2 Phosphorylation, and Rac Activation)-Human dermal microvascular endothelial cells (Emory Skin Disease Research Center) were cultured in 24-well plates with 10,000 cells/well for 24 h. Plates were washed by PBS, and 0.5 ml of fresh microvascular endothelial cell medium (18) with 0, 1, 6, or 9 g/ml honokiol was added. Cells were incubated for 30 min, and 30 g of TRAIL antibody/well (Alexis 804 -296-C100) or 30 g of isotype IgG control antibody (sc-2050, Santa Cruz Biotechnology) was added to the control plate according to the method of Clarke et al (19).…”

Capillarization of Hepatic Sinusoid by Liver Endothelial Cell-Reactive Autoantibodies in Patients with Cirrhosis and Chronic Hepatitis

“…16 In short, all identifiable vascular structures were excised from the liver specimen. Liver tissue was mechanically disrupted (into small cubes) and enzymatically digested with dispase (1.6 units/ml) overnight at 4°C.…”