Isolation and Characterization of Fetus Human Retinal Microvascular Endothelial Cells

Abstract

Purpose: To isolate and characterize primary fetus retinal microvascular endothelial cells (RMECs) in order to facilitate the study of their properties in vitro. Methods: Human RMECs were isolated from abortive fetuses whose gestational ages were between 20 and 28 weeks by mechanical morcel and trypsin-collagenase digestion, plated on fibronectin-coated wells and purified gradually in high-selective condition. The RMECs were characterized for expression and localization of endothelial cell markers by indirect immunocytochemical staining of von Willebrand factor (vWF) and CD34, the distribution of cell markers was observed by confocal immunofluorescence microscope. The ability of these cells to form capillary-like networks was assessed subsequently. Cobalt dichloride (CoCl₂) was used to simulate hypoxia in the culture condition and Western blot was used to detect the alternation of VEGF protein level under normoxia and hypoxia. Results: Isolation of fetus RMECs has been very difficult and primary culture of these special premature RMECs has not been previously reported due to many reasons. Here we established a simple and convenient method for the primary culture of fetus human RMECs. Our results show that delicate RMECs could be obtained readily and grow very fast, and the purity of RMECs was up to 95% demonstrated by specific staining for vWF in the cytoplasm. Our research also indicates that nearly 100% of cells could express CD34. The cells were successfully passaged and maintained in culture for 5–7 passages without a significant loss in expression of endothelia cell markers. The fetus RMECs formed capillary-like networks on the Matrigel well. Expression of VEGF protein increased significantly under hypoxia. Conclusions: Pure fetus RMECs could be obtained rapidly and easily by our method.Fetus RMECs have some specific properties. Hypoxia could induce expression of VEGF. Cell properties related with age of the donors should be considered while studying in vitro, especially in some age-specific diseases such as retinopathy of prematurity and diabetic retinopathy.