Biochemical and Immunocytochemical Characterization of Canine Corneal Cells Cultured in Two Different Media
Schorling, Jamie J
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The study purpose was to determine whether canine corneal cultures demonstrate superior growth when cultured in a fully defined epithelial selective medium, EpilifeÂ®, compared to Dulbeccoâ s modification of Eagleâ 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 EpilifeÂ®. 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 EpilifeÂ®. Stromal tissue segments in EpilifeÂ® 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 EpilifeÂ® compared to epithelial cells in DMEM with FBS or the stromal cells. Based on PCR results, EpilifeÂ® medium may support retention of the epithelial characteristic of CK5 mRNA expression better than DMEM with FBS.
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