Harvest and storage of adult human photoreceptor cells: the vibratome compared to the excimer laser.
OBJECTIVE
To develop a method using the vibratome and the excimer laser to harvest a sheet of human photoreceptor cells from the retinas of cadaveric donors.


METHODS
Adult human photoreceptor cells were harvested as intact sheets from the retinas of cadaver eyes using a vibratome or excimer laser. The sheets were embedded in 50% gelatin (in minimum essential medium and 300 mM sucrose) and stored at 4 degrees C. The morphology, integrity, viability and sterility of the harvested photoreceptor cells was studied.


RESULTS
Light and scanning electron microscopy demonstrated sheets of adult human photoreceptor cells with an outer nuclear layer and inner and outer segments with either method of harvest. The initial viability of the outer nuclear layer, harvested an average of 28.2 h after death, was > or =94.7%. Sheets stored up to 72 h after harvest maintained a viability of > or =86.5%. The sheet of cells harvested with the vibratome frequently fragmented (n = 25, 35%) during passage through the delivery cannula in contrast to the excimer laser. Harvested sheets were sterile when the gelatin powder was irradiated prior to reconstitution.


CONCLUSIONS
Intact, viable adult human photoreceptor cell sheets can be isolated from the retina of a cadaver using either the vibratome or the excimer laser and stored up to 72 h at 4 degrees C. With the vibratome, there is damage to the outer segments of the photoreceptors, the sheets are fragile, and the harvest of specimens is time-consuming as only one or two specimens can be harvested from a single donor retina. These technical limitations are avoided with the excimer laser.
Tezel
T H
TH
Department of Ophthalmology and Visual Sciences, Washington University School of Medicine, St. Louis, MO 63110, USA.
Kaplan
H J
HJ
D002102:Cadaver; D002470:Cell Survival / Q000502:physiology; D004950:Esterases / Q000378:metabolism; D006801:Humans; D007834:Lasers; D008855:Microscopy, Electron, Scanning; D010786:Photoreceptor Cells / Q000201:enzymology / Q000382:microbiology / Q000648:ultrastructure; D011309:Preservation, Biological; D013048:Specimen Handling / Q000295:instrumentation* / Q000379:methods*
D004950:Esterases