Change of Morphological and Functional Characteristics of Retinal Pigment Epithelium Cells during Cultivation of Retinal Pigment Epithelium-Choroid Perfusion Tissue Culture

Abstract

Aims: To evaluate the changes of morphological and functional characteristics of the retinal pigment epithelium (RPE)-choroid perfusion culture during cultivation. Methods: PorcineRPE-choroid tissue was cultivated in a perfusion tissue culture system. After the indicated times, histology, immunolocalization of collagen IV and von Willebrand factor, RPE cell viability with calcein-AM, TUNEL assay and occludin immunolocalization of RPE cells were examined. The tissue was treated with selective RPE treatment laser after different time periods and the wound healing response was characterized. Vascular endothelial growth factor secretion was measured by enzyme-linked immunosorbent assay. Results: On day 8, prominent morphological degenerative changes of RPE cells were observed in histology. According to the immunohistochemistry for collagen IV, the Bruch’s membrane did not display any obvious decomposition until day 8. Von Willebrand factor staining decreased during cultivation, especially at the choriocapillaris. Calcein-AM staining and TUNEL assay displayed the increase of apoptotic changes in only a minority of the cells on day 4, but in many cells on day 8. Occludin delocalization was observed on day 8. Selective RPE treatment laser-produced wounds were completely closed by monolayer RPE when wounded on fresh and 3-day-old cultures, but not when wounded on 6-day-old cultures. Vascular endothelial growth factor secretion was stable between days 2 and 5, but increased after that. Conclusion: Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance.

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

“…A constant perfusion generates a steady state equilibrium. RPE cells in this culture maintain their morphology and differentiation for the time period investigated [31].…”

“…Organ culture was prepared as previously described [31]. In brief, freshly slaughtered pig eyes were cleaned of adjacent tissue and immersed briefly in antiseptic solution.…”

Nicotine reduces VEGF-secretion and phagocytotic activity in porcine RPE

“…In our study, the secretion of VEGF in untreated cultures increases over time. As the tissue deteriorates during cultivation, the induction of VEGF-A is most likely a survival factor which is induced as the organ culture ages [26]. VEGF-A has recently been shown to be an autocrine survival factor for RPE cells [27], where the inhibition of VEGF or VEGF-R2 signaling results in a higher susceptibility towards oxidative stress.…”

Epidermal growth factor: the driving force in initiation of RPE cell proliferation

“…Nevertheless, several studies in the field of RPE cell biology verify the fact that ideal cell culture conditions are essential to maintain RPE-specific phenotype [16, 17]. Short incubation times enhance RPE cell quality for feasible rejuvenation and subsequent autologous transplantation of RPE cells [18].…”