Corneal organ culture: effects of serum and a stabilised form of L-glutamine.
OBJECTIVE
Organ culture medium for corneas contains labile components, such as L-glutamine, whose loss could be a limiting factor to the length of storage. The medium is also supplemented with fetal bovine serum (FBS), which can vary significantly between different batches. The aim of this study was to establish the need for FBS during corneal organ culture, and to determine whether substitution of L-glutamine by the stable dipeptide L-analyl-L-glutamine was beneficial.


METHODS
Porcine corneoscleral discs were suspended in 80 ml of organ culture medium (HEPES buffered Eagle's MEM with Earle's salts, 26 mmol/l NaHCO3, penicillin, streptomycin, and amphotericin B) and kept at 34 degrees C. The medium contained either 2 mmol/l L-glutamine or 2 mmol/l L-analyl-L-glutamine, and was either serum free or contained 2% FBS. At weekly intervals, five corneas from each group were stained with trypan blue and alizarin red S, and the surface area and shape of 100 endothelial cells were determined for each cornea.


RESULTS
No differences were observed between corneas in organ culture medium with L-glutamine or L-analyl-L-glutamine. In serum free medium, endothelial cell density remained constant for the first week, but then declined rapidly over the next 2 weeks. With 2% FBS, there was no loss of endothelial cells for the first 2 weeks, but cell density had halved by the fourth week of organ culture.


CONCLUSIONS
The presence of 2% FBS extended the period of endothelial stability, but no advantage was gained from the stabilised form of L-glutamine. The overall loss of endothelial cells was much greater than would be expected for human corneas.
Ayoubi
M G
MG
Department of Ophthalmology, University of Bristol.
Armitage
W J
WJ
Easty
D L
DL
D000818:Animals; D002417:Cattle; D002478:Cells, Cultured; D003470:Culture Media; D004728:Endothelium, Corneal / Q000166:cytology* / Q000187:drug effects; D005312:Fetal Blood / Q000502:physiology*; D005973:Glutamine / Q000031:analogs & derivatives / Q000494:pharmacology*; D009924:Organ Culture Techniques; D013552:Swine
D003470:Culture Media; D005973:Glutamine