title: Identification and evaluation of a thinning agent compatible with MegaCell DCS, an animal product-free corneal storage medium.
abstract: OBJECTIVE
  MegaCell 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.
  
  
  METHODS
  Corneas 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.
  
  
  RESULTS
  The 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.
  
  
  CONCLUSIONS
  PEG 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.
authors.affiliation: Academic Unit of Ophthalmology, University of Bristol, Bristol Eye Hospital, Lower Maudlin Street, Bristol, BS1 2LX, UK. Val2012Smith@gmail.com
authors.forename: Valerie A
authors.initials: VA
authors.lastname: Smith
authors.forename: Terrell K
authors.initials: TK
authors.lastname: Johnson
mesh_terms: D000199:Actins / Q000378:metabolism; D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D002452:Cell Count; D049109:Cell Proliferation; D003315:Cornea; D055954:Corneal Endothelial Cell Loss / Q000517:prevention & control; D003319:Corneal Stroma / Q000187:drug effects / Q000378:metabolism; D016895:Culture Media, Serum-Free / Q000737:chemistry / Q000494:pharmacology*; D003911:Dextrans / Q000737:chemistry; D004728:Endothelium, Corneal / Q000187:drug effects / Q000473:pathology; D005260:Female; D006801:Humans; D008297:Male; D008875:Middle Aged; D009924:Organ Culture Techniques; D009926:Organ Preservation; D019188:Organ Preservation Solutions / Q000737:chemistry / Q000494:pharmacology*; D020442:Poloxamer / Q000737:chemistry; D011092:Polyethylene Glycols / Q000737:chemistry
chemical_list: C541116:ACTA2 protein, human; D000199:Actins; D016895:Culture Media, Serum-Free; D003911:Dextrans; D019188:Organ Preservation Solutions; D020442:Poloxamer; D011092:Polyethylene Glycols