title: Novel primary epithelial cell toxicity assay using porcine corneal explants.
abstract: OBJECTIVE
  The purpose of this study was to develop a novel primary epithelial cell toxicity assay using porcine corneal explant and evaluate the assay using benzalkonium chloride (BAK).
  
  
  METHODS
  Circular corneal explants were trephined from the peripheral cornea of porcine eyes using 2-mm biopsy punches, and placed on 6-well culture dishes with a culture medium. After incubation for 12 hours, 50 μL of BAK at 0.00001%, 0.0001%, 0.001%, or 0.01% was applied to each well for 2 minutes. After washing, explants were cultured for another 24 hours, then epithelial outgrowth was photographed and measured. Corneal immunohistochemical characteristics were evaluated by cytokeratin (CK) 3, CK12, and ZO-1. Cell toxicity was evaluated by WST-8 assay, Ki-67 staining, and TUNEL assay.
  
  
  RESULTS
  Epithelial cells migrated outward concentrically from the corneal explant as time elapsed and were positive for CK3, CK12, and ZO-1. The outgrowth rate decreased significantly with 0.0001%, 0.001%, and 0.01% BAK compared with the phosphate-buffered saline (PBS) control (P < 0.01), and the decrease was BAK concentration dependent. Numbers of viable cells and Ki-67-positive cells also decreased significantly with 0.01% BAK compared with the PBS control (P < 0.05). TUNEL-positive cells were present in the epithelial outgrowth. Moreover, TUNEL-positive cell density tended to increase with 0.01% BAK compared with the PBS control (P = 0.14) and 0.00001% BAK (P = 0.081).
  
  
  CONCLUSIONS
  Our novel toxicity assay using porcine corneal explant is simple and reflects the effect of single and short-duration instillation of eye drops. The method is useful for evaluation of corneal toxicity at low concentrations of BAK.
authors: *Department of Ophthalmology, Tokyo Medical University, Tokyo, Japan; †Department of Surgery, Keio University, Tokyo, Japan; and ‡Veterinary Medical Center, Faculty of Agriculture, Tottori University, Tottori, Japan.
authors: Hiroki
authors: H
authors: Takahashi
authors: Kazuki
authors: K
authors: Tajima
authors: Takaaki
authors: T
authors: Hattori
authors: Naoyuki
authors: N
authors: Yamakawa
authors: Norihiko
authors: N
authors: Ito
authors: Hiroshi
authors: H
authors: Goto
mesh_terms: D000818:Animals; D001548:Benzalkonium Compounds / Q000633:toxicity*; D015415:Biomarkers / Q000378:metabolism; D002452:Cell Count; D002465:Cell Movement / Q000187:drug effects; D002478:Cells, Cultured; D019573:Epithelium, Corneal / Q000187:drug effects* / Q000378:metabolism; D020287:In Situ Nick-End Labeling; D053551:Keratin-12 / Q000378:metabolism; D053558:Keratin-3 / Q000378:metabolism; D019394:Ki-67 Antigen / Q000378:metabolism; D009924:Organ Culture Techniques; D011310:Preservatives, Pharmaceutical / Q000633:toxicity*; D013552:Swine; D013778:Tetrazolium Salts / Q000378:metabolism; D018675:Toxicity Tests; D062826:Zonula Occludens-1 Protein / Q000378:metabolism
chemical_list: C476329:2-(2-methoxy-4-nitrophenyl)-3-(4-nitrophenyl)-5-(2,4-disulfophenyl)-2H tetrazolium monosodium salt; D001548:Benzalkonium Compounds; D015415:Biomarkers; D053551:Keratin-12; D053558:Keratin-3; D019394:Ki-67 Antigen; D011310:Preservatives, Pharmaceutical; D013778:Tetrazolium Salts; D062826:Zonula Occludens-1 Protein