Identification and evaluation of a thinning agent compatible with MegaCell DCS, an animal product-free corneal storage medium

Abstract

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.

Serum-free medium and hydroxyethyl starch supports cell survival better than Minimal Essential Medium and dextran in organ-cultured mouse corneas

“…It was already described that deswelling agents like dextran may contribute to metabolic changes in donor corneas and to corneal endothelial cell loss 11 15 16. In our study, incubation in SFM-HES for 4 days still is favourable to a 1-day incubation in MEM-dextran or a 4-day incubation in MEM-HES.…”

Deformation velocity imaging using optical coherence tomography and its applications to the cornea

“…To counteract this, donor corneas are “de-swelled” before use in a hypertonic solution, usually containing dextran, which removes the excess water from the cornea by osmosis. The health of the endothelial layer affects the de-swelling rate [ 19 ] and conversely, the de-swelling process can damage the health of the donor tissue [ 20 ]. For these reasons there has been substantial research to optimise the de-swelling time [ 9 , 21 ], improve the method [ 10 ], or use alternative osmotic agents such as poloxamers or PEG [ 11 , 12 ].…”

Fetal bovine serum (FBS): Past – present – future

“…The organ culture media contain 2 to 8% FBS (Doughman et al, 2012). A few approaches report the use of alternative media in organ or tissue storage, but none of these were brought to a new standard recommendation (Rieck et al, 2003;Smith and Johnson, 2012;Yokoo et al, 2008). In fact, all chemically-defined non-serum alternatives tested to date do not show a better endothelial cell survival under long-term storage conditions.…”