Cultivation of the Human Choroidal Microvascular Endothelial Cells by Dynabeads CD31 and Measurement of Their Growth Curve by MTT

Abstract

Abstract: : Purpose: To establish a rapid cell culture method of human choroidal endothelial cells (HCMECs) in order to further study a variety of chorioretinal diseases which involves the HCMECs. Additionally, to study the growing states of HCMECs and to provide theoretical basis for the in vitro experiments on HCMECs. Methods: The human choroidal tissues were digested in two steps by trypsin and collagenase combined with the purification technique of CD31 Dynabeads. The obtained HCMECs were primarily cultured and subcultured. The cultured HCMECs were confirmed with the morphologic observation under the inverted phase contrast microscopy, transmission electron microscopy, and immunohistochemical staining of the FVIII–Ag, CD31, CD34.MTT operating curve and HCMECs growth curve were determined. Results: Sufficient number of HCMECs with cluster appearance was obtained after digestion and purification. In primary culture, HCMECs appeared long and thin at initial stage and became polygonal, or round when they grew into sheets. HCMECs maintained cobblestone morphology after HCMECs nests became confluent. In about 2–3 weeks HCMECs could grow to cover the whole dish floor and form a monolayer. After a long period of confluent state, contact inhibition started. A lot of dead HCMECs were observed on the following day. The survived HCMECs remained polygonal, or round. Cobblestone appearance was seen after HCMECs nests became confluent again. In about 1 week the subculture occurred again after pre–subculture. The Weibel–Palade body which is the characteristic marker of endothelial cells was seen by transmission electron microscopy. More than 95% of cultured HCMECs were positive for the FVIII–Ag, CD31, CD34 immunohistochemical stain,those in negative control group did not stain. The growth curve of HCMECs measured by MTT colorimetry showed that after passage, the activity of cells was weakened on day 1. The cells lived through 2–3 days of latency, followed by the heterogonous stage, and the flat stage (day 5 to 8). On day 9, the inhibition of cell growth happened. Conclusions: Our experiment was successful in establishing a rapid method to culture HCMECs. The method is easy to apply for obtaining sufficient number of highly purified HCMECs. HCMECs cultured in vitro are capable of proliferating under proper conditions, but this capability is limited.

Keywords: choroid • vascular cells • microscopy: electron microscopy