Change of morphological and functional characteristics of retinal pigment epithelium cells during cultivation of retinal pigment epithelium-choroid perfusion tissue culture.
OBJECTIVE
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.


CONCLUSIONS
Under the stated culture perfusion conditions, porcine RPE-choroid tissue was suitable for experimentation up to 5 days of maintenance.
Miura
Yoko
Y
Department of Ophthalmology, University of Kiel, Kiel, Germany.
Klettner
Alexa
A
Noelle
Bernhard
B
Hasselbach
Heike
H
Roider
Johann
J
D000818:Animals; D017209:Apoptosis; D016570:Bruch Membrane / Q000473:pathology; D002470:Cell Survival; D002829:Choroid / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D024141:Collagen Type IV / Q000378:metabolism; D004797:Enzyme-Linked Immunosorbent Assay; D005452:Fluoresceins / Q000378:metabolism; D007124:Immunoenzyme Techniques; D020287:In Situ Nick-End Labeling; D053685:Laser Therapy; D008565:Membrane Proteins / Q000378:metabolism; D062793:Occludin; D009924:Organ Culture Techniques; D055213:Retinal Pigment Epithelium / Q000378:metabolism / Q000473:pathology* / Q000601:surgery; D013552:Swine; D013997:Time Factors; D042461:Vascular Endothelial Growth Factor A / Q000378:metabolism; D014945:Wound Healing; D014841:von Willebrand Factor / Q000378:metabolism
D024141:Collagen Type IV; D005452:Fluoresceins; D008565:Membrane Proteins; D062793:Occludin; D042461:Vascular Endothelial Growth Factor A; D014841:von Willebrand Factor; C085925:calcein AM