Presentation Title

Conditionally Immortalized Retinal Capillary Endothelial Cells

Format

Event

Start Date

12-2-2010 12:00 AM

Abstract

Purpose. Human retinal capillary endothelial cells (hRCE) may play an important role in the early phases of bacterial endophthalmitis. A limited in vitro life span, and low endothelial cell yields severely restricts investigation of the properties of these cells. Therefore, we sought to increase the number of available cells by immortalization. Methods. Retinas from human donor eyes were dissected from the optic nerve and digested with 0.25% collagenase. Vessel fragments were captured on a 20μ mesh and plated. Endothelial cells were selected using CD31 coated beads. Immortalization of primary hRCE and human aortic endothelial cells (HAEC) was performed using an amphotrophic replication-defective retroviral construct (pLXSN16E6/E7). Immunocytochemistry (ICC) was used to detect endothelial markers (vWF, CD31, CD62E) on primary and presumptively immortalized cells. HAEC served as positive EC controls. ELISA was also performed on the presumed immortalized cells to confirm expression of CD54, CD31 and CD62E. Results. Using ICC, primary hRCE (3 patients) expressed vWF and CD31. The staining intensities were similar to that seen in HAEC (1 patient). Primary hRCE and HAEC also responded to TNF-alpha stimulation as measured by CD62E expression. Treating cells with pLXSN16E6/E7 increased the time in culture of primary hRCE from a maximum 5 passages to at least 12 passages and HAEC from 10 to at least 20 passages. ICC of both hRCE and HAEC treated with pLXSN16E6/E7 showed similar EC marker profiles. ELISA on virus treated hRCE and HAEC confirmed expression of CD54, CD31 and CD62E. Conclusion. We have extended the life span of primary hRCE and these conditionally immortalized cells appear to maintain their phenotype for the characteristics examined. This technique will allow examination of their contribution to bacterial endophthalmitis in vitro. Commercial Relationship. M.J. Giese, None; S.A. Rayner, None; B.J. Mondino, None. Grants. The Card Family Research Fund, Research to Prevent Blindness Senior Scientific Investigator Award.

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Feb 12th, 12:00 AM

Conditionally Immortalized Retinal Capillary Endothelial Cells

Purpose. Human retinal capillary endothelial cells (hRCE) may play an important role in the early phases of bacterial endophthalmitis. A limited in vitro life span, and low endothelial cell yields severely restricts investigation of the properties of these cells. Therefore, we sought to increase the number of available cells by immortalization. Methods. Retinas from human donor eyes were dissected from the optic nerve and digested with 0.25% collagenase. Vessel fragments were captured on a 20μ mesh and plated. Endothelial cells were selected using CD31 coated beads. Immortalization of primary hRCE and human aortic endothelial cells (HAEC) was performed using an amphotrophic replication-defective retroviral construct (pLXSN16E6/E7). Immunocytochemistry (ICC) was used to detect endothelial markers (vWF, CD31, CD62E) on primary and presumptively immortalized cells. HAEC served as positive EC controls. ELISA was also performed on the presumed immortalized cells to confirm expression of CD54, CD31 and CD62E. Results. Using ICC, primary hRCE (3 patients) expressed vWF and CD31. The staining intensities were similar to that seen in HAEC (1 patient). Primary hRCE and HAEC also responded to TNF-alpha stimulation as measured by CD62E expression. Treating cells with pLXSN16E6/E7 increased the time in culture of primary hRCE from a maximum 5 passages to at least 12 passages and HAEC from 10 to at least 20 passages. ICC of both hRCE and HAEC treated with pLXSN16E6/E7 showed similar EC marker profiles. ELISA on virus treated hRCE and HAEC confirmed expression of CD54, CD31 and CD62E. Conclusion. We have extended the life span of primary hRCE and these conditionally immortalized cells appear to maintain their phenotype for the characteristics examined. This technique will allow examination of their contribution to bacterial endophthalmitis in vitro. Commercial Relationship. M.J. Giese, None; S.A. Rayner, None; B.J. Mondino, None. Grants. The Card Family Research Fund, Research to Prevent Blindness Senior Scientific Investigator Award.