Purpose: Gibco’s Minimum Essential Medium with Earle’s salts and HEPES supplemented with glutamine, antibiotics (EB MEM) and 2% foetal calf serum (FCS) is used in European eye banks to store corneas. Although FCS is important to endothelial cell survival in this medium, it is a potential biohazard. Megacell MEM, formulated to reduce the FCS requirement of cells by a factor of 5, has therefore been evaluated as a corneal storage medium. Methods: Corneal stromal and epithelial cells were incubated in Megacell MEM (serum-free or 2% FCS) to assess their viability in these media. Endothelial cell densities of paired corneas held in either EB MEM 2% FCS or Megacell MEM (serum-free or 2% FCS) were measured over 5 weeks. Discs subsequently punched from the centre of these corneas were weighed, dried and reweighed to determine hydration levels. Results: Both corneal stromal and epithelial cells proliferated in Megacell MEM 2% FCS. Relative to EB MEM, 2% FCS Megacell MEM prolonged the viability of corneal endothelial cells and improved their morphological appearance, irrespective of whether it contained FCS or not. This was independent of corneal swelling. Conclusion: Serum-free Megacell MEM is a better storage medium than EB MEM 2% FCS for corneas destined for transplantation

Purpose: The traditional medium for storing corneas in European Eye Banks is Gibco’s MEM (Eagle’s) with Earle’s salts and Hepes containing 2% fetal calf serum, glutamine and antimycotics. Although serum-free MegaCell™ MEM has been reported to be more suitable for this purpose, it contains components of animal origin that potentially pose health risks to corneal recipients. The possibility of removing or replacing these components has therefore been investigated. Methods: A MegaCell basal medium (DME) and a formulation of this (MegaCell DCS), which contains no components of animal origin, have been prepared. The viability of the endothelial, epithelial and stromal cells of corneas held in these media has been assessed, their stress levels monitored and water content determined. Results: The endothelial cell count and morphology of corneas held in MegaCell DME and DCS for 30 days remained little changed. Their epithelial and stromal cells also retained their ability to proliferate in culture. Neither DME nor DCS prevented corneal swelling but the lack of endogenous MMP-2 activation indicated that the corneas were not subject to metabolic stress. Conclusion: MegaCell DCS is an animal product-free medium formulated for corneal storage. The quality of corneas held in this medium is similar to those held in MegaCell MEM.

PurposeMegaCell DCS, an animal product-free culture medium formulated for storing corneas, is superior to the traditionally used MEM (Eagle’s) with Earles salts, Hepes, and supplemented with foetal calf serum (2 %), glutamine and an antibiotic cocktail (EB MEM). Because this medium does not prevent corneal swelling, and Dextran T500, which is traditionally used for reversing this process before transplant may have adverse effects on corneas, the purpose of the current investigation was to identify an alternative polymer that is compatible with MegaCell DCS.MethodsCorneas maintained in MegaCell DCS or EB MEM were transferred to either EB MEM 5 % Dextran T500 or MegaCell DCS containing 5 % Dextran T500, 4 % polyethylene glycol (PEG) 10,000, PEG 35,000 (2 %, 3 %, 4 %) or Poloxamer 188 (4 %). Endothelial cell losses were determined and corneal hydration levels measured. Stromal cell cultures were generated and immunostained with anti α-SMA antibody. Janus Green was used to compare the viability of endothelial cells of corneas maintained in MegaCell DCS and EB MEM and respectively thinned with PEG 35,000 and Dextran T500.ResultsThe rates of endothelial cell loss from corneas held in MegaCell DCS and thinned in MegaCell DCS containing 5 % Dextran T500, 4 % PEG 10,000 and 4 % Poloxamer 188 for 6 days were similar. When explants of these corneas were cultured myofibroblasts were generated. Although at concentrations of 4 % (w/v) both PEG 10,000 and Poloxamer 188 caused excessive dehydration, the hydration levels of corneas held in MegaCell DCS containing 3 % PEG 35,000 were similar to those of corneas held in EB MEM 5 % Dextran T500. Endothelial cell losses after 6 days were negligible, explants of the corneas generated uniform fibroblastic stromal cell cultures and the extents of Janus Green staining were similar. Over 20 days the inclusion of 5 % Dextran T500 in EB MEM but not 3 % PEG 35,000 in MegaCell DCS, increased the rate of endothelial cell loss.ConclusionPEG 35,000 at a concentration of 3 % w/v does not induce endothelial cell loss and is compatible with MegaCell DCS for thinning corneas prior to transplantation.