Biochemical and Immunocytochemical Characterization of Canine Corneal Cells Cultured in Two Different Media

The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, Epilife®, compared to Dulbecco's modification of Eagle's medium (DMEM) with fetal bovine serum (FBS), and to characterize cultured canine corneal cells. Superficial keratectomies were performed on three dogs. Samples were trypsinized to separate cell layers. Post-trypsinization, immunohistochemistry confirmed that epithelial cells had been released from the stroma. Both cell populations (presumed epithelial cells and stromal tissues) were cultured in DMEM with FBS or Epilife®. First passage cells were fixed for immunocytochemistry and prepared for PCR. Immunocytochemical staining for pancytokeratin, vimentin, and E-cadherin was evaluated, and immunofluorescence for zonula occludens-1 was attempted. Amplification of cytokeratin 5 (CK5) mRNA was assessed by PCR. Primary presumed epithelial cells grew faster when cultured in DMEM with FBS compared to Epilife®. Stromal tissue segments in Epilife® medium failed to adhere to culture plates, indicating that this medium may inhibit attachment and growth of non-epithelial tissues. Staining of corneal tissue segments confirmed that epithelial layers were pancytokeratin and E-cadherin positive, while stromal cells were vimentin positive. Immunocytochemistry of cultured cells revealed that epithelial cells stained positively for pancytokeratin, vimentin, and E-cadherin, while stromal cells remained only vimentin positive. Greater amplification of CK5 mRNA occurred from epithelial cells grown in Epilife® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, Epilife® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS. Master of Science