title: Implications for Ophthalmic Formulations: Ocular Buffers Show Varied Cytotoxic Impact on Human Corneal-Limbal and Human Conjunctival Epithelial Cells.
abstract: OBJECTIVE
  To investigate toxicity associated with buffers commonly used in topical ocular drug formulations using a human corneal-limbal epithelial (HCLE) and a human conjunctival epithelial (HCjE) cell model.
  
  
  METHODS
  HCLE and HCjE cells were incubated for 10, 30, or 60 minutes with 4 different buffers based on borate, citrate, phosphate, and Tris-HCl at 10, 50, and 100 mM concentrations. To detect possible delayed effects on cell viability, after 60 minutes of buffer incubation, cells were further incubated for 24 hours with a cell medium. Cell viability was determined using a colorimetric XTT-based assay. The morphology of cells was also investigated.
  
  
  RESULTS
  HCjE cells showed more sensitivity to buffer incubation than HCLE cells. The 100 mM phosphate buffer displayed significant delayed effects on cell viability of HCLE 16.8 ± 4.8% and HCjE 39.2 ± 6.1% cells after 60 minutes of exposure (P < 0.05). HCjE cell viability was reduced after 60 minutes incubations with 50 and 100 mM citrate buffer to 42.8 ± 6.5% and 39.3 ± 7.9%, respectively, and even lower percentages at the delayed time point (both P < 0.05). HCLE cell morphology was distinctly altered by 100 mM phosphate and Tris buffers after 30 minutes, whereas HCjE cells already showed marked changes after 10 minutes of exposure to 100 mM citrate and phosphate buffers.
  
  
  CONCLUSIONS
  We observed a time-dependent decrease of viability in both HCLE and HCjE cells exposed to higher buffer concentrations. Therefore, we propose further in vivo studies to translate these finding to humans to discern the real effects of the buffer concentration in eye drops on the ocular surface.
authors.affiliation: *OCUVAC-Center of Ocular Inflammation and Infection, Laura Bassi Centers of Expertise, Center for Pathophysiology, Infectiology and Immunology, Medical University of Vienna, Vienna, Austria; and †Croma-Pharma Gesellschaft m.b.H., Leobendorf, Austria.
authors.forename: Nadine
authors.initials: N
authors.lastname: Schuerer
authors.forename: Elisabeth
authors.initials: E
authors.lastname: Stein
authors.forename: Aleksandra
authors.initials: A
authors.lastname: Inic-Kanada
authors.forename: Marion
authors.initials: M
authors.lastname: Pucher
authors.forename: Christine
authors.initials: C
authors.lastname: Hohenadl
authors.forename: Nora
authors.initials: N
authors.lastname: Bintner
authors.forename: Ehsan
authors.initials: E
authors.lastname: Ghasemian
authors.forename: Jacqueline
authors.initials: J
authors.lastname: Montanaro
authors.forename: Talin
authors.initials: T
authors.lastname: Barisani-Asenbauer
mesh_terms: D002021:Buffers; D002460:Cell Line; D002470:Cell Survival / Q000187:drug effects; D003228:Conjunctiva / Q000166:cytology*; D004847:Epithelial Cells / Q000187:drug effects*; D006801:Humans; D016850:Limbus Corneae / Q000166:cytology*; D009883:Ophthalmic Solutions / Q000737:chemistry / Q000633:toxicity*; D004364:Pharmaceutical Preparations / Q000737:chemistry; D013997:Time Factors
chemical_list: D002021:Buffers; D009883:Ophthalmic Solutions; D004364:Pharmaceutical Preparations