title: Evaluation of potential organ culture media for eye banking using human donor corneas.
abstract: OBJECTIVE
  To evaluate the ability of different commercially available cell culture solutions to preserve human donor corneas during 3 weeks of "closed system" organ culture at physiological temperature. This screening was performed in an attempt to establish a rational basis for the development of a serum-free organ culture medium for eye banking.
  
  
  METHODS
  72 normal human donor corneas were organ cultured for 21 days at 31 degrees C in eight different test media (nine corneas in each group). The basic culture solutions included: minimal essential medium (MEM), MEM with stabilised L-glutamine, M199, DIF-1000, SFM, F99, and F99 with ascorbic acid, insulin, bFGF, transferrin, selenium, and lipids (termed F99-Sr). All media were supplemented with 2% fetal calf serum (FCS), except for MEM, which was also studied at 8% FCS. The evaluation parameters included: (1) the endothelial cell loss as evaluated using trypan blue staining; (2) the ability of keratocytes and endothelial cells to incorporate tritiated uridine into RNA as evaluated using autoradiography and digital image analysis; (3) the leakage of immunogenic keratan sulphate as assessed using ELISA; and (4) changes in storage medium pH, glucose, and lactate content.
  
  
  RESULTS
  SFM induced the lowest endothelial cell loss of 14% (SD 2%) and the highest RNA synthesis rates of all test solutions supplemented with 2% FCS. Corneas stored in SFM also showed the least leakage of keratan sulphate and the highest glucose consumption and lactate production. In five media (MEM with 2% FCS, MEM with stabilised L-glutamine, M199, F99, and F99-Sr), comparable and intermediate potentials for organ culture were observed with endothelial cell loss of 16-19%. By contrast, 29% (4%) of the endothelium was lost after storage in DIF-1000. Interestingly, the use of 8% FCS (in MEM) had a marked protective effect on the endothelium, which showed the highest RNA synthetic activity combined with a cell loss of only 11% (4%), compared with 19% (6%) at 2% FCS (p<0.05).
  
  
  CONCLUSIONS
  Among the present test solutions, SFM appears to be the most prominent candidate for a new corneal organ culture medium and should be further tested and possibly refined to effectively substitute serum addition.
authors.affiliation: Department of Ophthalmology, Arhus University Hospital, 8000 Arhus C, Denmark. tmp@dadlnet.dk
authors.forename: T
authors.initials: T
authors.lastname: Møller-Pedersen
authors.forename: U
authors.initials: U
authors.lastname: Hartmann
authors.forename: H J
authors.initials: HJ
authors.lastname: Møller
authors.forename: N
authors.initials: N
authors.lastname: Ehlers
authors.forename: K
authors.initials: K
authors.lastname: Engelmann
mesh_terms: D000328:Adult; D000368:Aged; D000369:Aged, 80 and over; D016923:Cell Death / Q000187:drug effects; D003315:Cornea / Q000166:cytology / Q000187:drug effects* / Q000378:metabolism; D016039:Corneal Transplantation; D003470:Culture Media / Q000378:metabolism / Q000494:pharmacology*; D016895:Culture Media, Serum-Free; D004728:Endothelium, Corneal / Q000166:cytology / Q000187:drug effects / Q000378:metabolism; D005125:Eye Banks; D006801:Humans; D007632:Keratan Sulfate / Q000378:metabolism; D008875:Middle Aged; D009924:Organ Culture Techniques / Q000379:methods; D012313:RNA / Q000096:biosynthesis; D014021:Tissue Preservation / Q000379:methods
chemical_list: D003470:Culture Media; D016895:Culture Media, Serum-Free; D012313:RNA; D007632:Keratan Sulfate