Development and characterization of a canine oral mucosa equivalent in a serum-free environment.
The objectives of this study were to develop a serum-free system for culturing canine oral keratinocytes, the construction and characterization of a canine ex vivo produced oral mucosa equivalent (EVPOME), and transduction green fluorescent protein (GFP) into keratinocytes as a post-grafting tracking marker. Dissociated canine buccal mucosa keratinocytes were cultured in a chemically defined serum-free medium, Epilife trade mark. First-passage keratinocytes were transfected with the GFP gene using a lentiviral vector, sorted by flow cytometer and seeded onto a dermal equivalent, AlloDerm(R) to form EVPOMEs. The EVPOME was characterized by histology and immunohistochemistry, for p63, Ki-67, and involucrin. Laser confocal microscopy was used to locate GFP-transfected keratinocytes within the EVPOME. Cultured canine oral keratinocytes grew rapidly over the first three passages and then the proliferative rate decreased. The canine EVPOME formed a well-stratified epithelial layer. The majority of p63 and Ki-67 immunopositive cells were located in the basal layer whereas cytoplasmic involucrin expression was seen in the suprabasal layers, similar to native canine buccal mucosa. Under laser confocal microscopy, significant green fluorescence was observed throughout the EVPOME. In conclusion, canine EVPOMEs were successfully fabricated in a defined serum-free system with similar characteristics to native buccal mucosa. GFP-transfected canine oral keratinocytes could be identified within the EVPOME.
Song
Junhui
J
Department of Oral and Maxillofacial Surgery, University of Michigan Medical Center, 1500 East Medical Center Drive, Ann Arbor, Michigan 48109-0018, USA.
Izumi
Kenji
K
Lanigan
Thomas
T
Feinberg
Stephen E
SE
D000818:Animals; D015415:Biomarkers; D049109:Cell Proliferation; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free; D004285:Dogs; D049452:Green Fluorescent Proteins / Q000235:genetics / Q000378:metabolism; D006801:Humans; D007150:Immunohistochemistry; D015603:Keratinocytes / Q000166:cytology / Q000502:physiology; D008422:Materials Testing; D009061:Mouth Mucosa / Q000033:anatomy & histology / Q000502:physiology*; D046509:Tissue Culture Techniques; D023822:Tissue Engineering / Q000379:methods
D015415:Biomarkers; D016895:Culture Media, Serum-Free; D049452:Green Fluorescent Proteins