Growth of cultured porcine retinal pigment epithelial cells

Abstract

ABSTRACT.Purpose: To establish and characterize cultures of porcine retinal pigment epithelial (pRPE) cells in order to produce confluent monolayers of cells for transplantation. Methods: Primary pRPE cell cultures were established. Cell morphology was assessed by phase contrast and electron microscopy. Growth was determined by the crystal violet dye uptake assay. DNA synthesis and content were measured by incorporation of 3 H-thymidine and flow cytometry. Results: This primary culture resulted in cells with well-preserved morphology that could be propagated in up to six passages. The deterioration observed over time in cultures was not due to a constant high rate of cell turnover as postconfluency cell proliferation was limited. However, a large fraction of the cells had a high DNA content despite a lack of active DNA synthesis. Conclusions: The present method yields pRPE cells of high purity and proliferative capacity with preserved epithelial phenotype. However, aberrant DNA profiles and the deterioration of cell morphology observed over time in this graft material represent serious problems in RPE transplantation.

The RhoA Activator GEF-H1/Lfc Is a Transforming Growth Factor-β Target Gene and Effector That Regulates α-Smooth Muscle Actin Expression and Cell Migration

“…Primary RPE cells in culture transdifferentiate into myofibroblast-like cells not only when treated with TGF-β but also spontaneously when plated at low density (Grisanti and Guidry, 1995; Lee et al , 2001; Wiencke et al , 2003). Transdifferentiated RPE cells up-regulate both α-SMA and GEF-H1, supporting a myofibroblast-like phenotype (Figure 1G).…”

“…In primary RPE cells in culture, transdifferentiation can be induced when cells are plated at low density, resulting in increased expression of α-SMA (Grisanti and Guidry, 1995; Lee et al , 2001; Wiencke et al , 2003) as well as GEF-H1 (Figure 3). As it has been suggested that at low confluence RPE cell secrete TGF-β, we inhibited the TGF-β receptor I with the ALK5 inhibitor and indeed found that it prevented morphological degeneration as well as α-SMA and GEF-H1 up-regulation.…”

Fucoidan Reduces Secretion and Expression of Vascular Endothelial Growth Factor in the Retinal Pigment Epithelium and Reduces Angiogenesis In Vitro

“…Primary porcine RPE cells are an established model and were isolated as previously described [29], [30]. The eyes were cleaned of adjacent tissue and immersed briefly in antiseptic solution.…”

“…The range of detection of the ELISA was between 15 pg/ml and 1046 pg/ml. The ELISA detects all isoforms of VEGF-A, and readily detects porcine VEGF-A [29] as well as human VEGF-A.…”

The Acta‐EVER Honorary Lecture 2007

“…In both fields of research, he supervised young PhD students, in what has become an extremely fruitful and successful career in academia. Already, Morten has supervised more than 10 PhD students and the following refreneces give some examples of this important work (Wiencke et al. 2003; Kiilgaard et al.…”