Abstract
AIMS: 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. CONCLUSION: 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.
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- Armitage W. J., Moss S. J., Easty D. L., Bradley B. A. Supply of corneal tissue in the United Kingdom. Br J Ophthalmol. 1990 Nov;74(11):685–687. doi: 10.1136/bjo.74.11.685. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Borderie V. M., Kantelip B. M., Delbosc B. Y., Oppermann M. T., Laroche L. Morphology, histology, and ultrastructure of human C31 organ-cultured corneas. Cornea. 1995 May;14(3):300–310. doi: 10.1097/00003226-199505000-00012. [DOI] [PubMed] [Google Scholar]
- Doughman D. J., Harris J. E., Schmitt M. K. Penetrating keratoplasty using 37 C organ cultured cornea. Trans Sect Ophthalmol Am Acad Ophthalmol Otolaryngol. 1976 Sep-Oct;81(5):778–793. [PubMed] [Google Scholar]
- Hjortdal J. O., Ehlers N., Andersen C. U. Some metabolic changes during human corneal organ culture. Acta Ophthalmol (Copenh) 1989 Jun;67(3):295–300. doi: 10.1111/j.1755-3768.1989.tb01874.x. [DOI] [PubMed] [Google Scholar]
- McDermott R. H., Butler M. Uptake of glutamate, not glutamine synthetase, regulates adaptation of mammalian cells to glutamine-free medium. J Cell Sci. 1993 Jan;104(Pt 1):51–58. doi: 10.1242/jcs.104.1.51. [DOI] [PubMed] [Google Scholar]
- Redmond R. M., Armitage W. J., Whittle J., Moss S. J., Easty D. L. Long-term survival of endothelium following transplantation of corneas stored by organ culture. Br J Ophthalmol. 1992 Aug;76(8):479–481. doi: 10.1136/bjo.76.8.479. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Spolarics Z., Lang C. H., Bagby G. J., Spitzer J. J. Glutamine and fatty acid oxidation are the main sources of energy for Kupffer and endothelial cells. Am J Physiol. 1991 Aug;261(2 Pt 1):G185–G190. doi: 10.1152/ajpgi.1991.261.2.G185. [DOI] [PubMed] [Google Scholar]
- Summerlin W. T., Miller G. E., Harris J. E., Good R. A. The organ-cultured cornea: an in vitro study. Invest Ophthalmol. 1973 Mar;12(3):176–180. [PubMed] [Google Scholar]
- Taylor M. J., Hunt C. J. Dual staining of corneal endothelium with trypan blue and alizarin red S: importance of pH for the dye-lake reaction. Br J Ophthalmol. 1981 Dec;65(12):815–819. doi: 10.1136/bjo.65.12.815. [DOI] [PMC free article] [PubMed] [Google Scholar]
- Vail A., Gore S. M., Bradley B. A., Easty D. L., Rogers C. A., Armitage W. J. Influence of donor and histocompatibility factors on corneal graft outcome. Transplantation. 1994 Dec 15;58(11):1210–1216. [PubMed] [Google Scholar]
- Vail A., Gore S. M., Bradley B. A., Easty D. L., Rogers C. A. Corneal graft survival and visual outcome. A multicenter Study. Corneal Transplant Follow-up Study Collaborators. Ophthalmology. 1994 Jan;101(1):120–127. doi: 10.1016/s0161-6420(94)31376-5. [DOI] [PubMed] [Google Scholar]
- Wu G., Greene L. W. Glutamine and glucose metabolism in bovine blood lymphocytes. Comp Biochem Physiol B. 1992 Dec;103(4):821–825. doi: 10.1016/0305-0491(92)90199-2. [DOI] [PubMed] [Google Scholar]