Adipose Tissue-derived Stem Cellssheets Fabricated In Vitro On Temperature-Responsive Scaffolds

[Purposes] Rabbit adipose tissue-derived stem cells (ADSCs) and oral mucosal epithelial cells (OMECs) were cultivated in vitro on temperature-responsive scaffolds (TRSs). We compared the biological characteristics between ADSCs and OMECs, so as to explore the possibility of ADSCs function as the stem cells for the ocular surface reconstruction.[Methods] N-isopropylacrylamide (IPAAm) was dissolved in isopropyl alcohol. IPAAm solution was added to each polystyrene tissue culture dish and then irradiated with an election beam. Rabbit adipose tissue and oral mucosal epithelial tissue were digested and centrifuged to obtain primary cells. The second-generation ADSCs were seeded at the TRSs, while primary-generation OMECs were inoculated with mitomycin C-treated3T3cells on TRSs. The comparisons between ADSCs and OMECs were based on the following evaluation items:(1) cell morphology,(2) cell growth rate,(3) cell sheet recovery,(4) total cell number,(5) degree of stratification (HE staining),(6)Immunohistochemical staining for anti-cytokeratin12(corneal and oral mucosal differentiated epithelial cell markers) and P63, ABCG2(makers of epithelial stem/progenitor),(7) scanning electron microscopy.[Results] ADSCs elongated fusiform shape while OMECs showed a cobblestone appearance.The cell growth cycle (coverage up to100%) of ADSCs and OMECs were12～14days and14～16days respectively. Averages of total cell number in ADSCs and OMECs sheets were(7.9±1.1)×105and(45.8±26.5)×105cells/sheet, respectively. After HE-staining, ADSCs sheet comprised of only one to three cell layers, while OMECs showed four to five cell layers. P63, ABCG2, CK12were expressed in both ADSCs and OMECs sheets.Scanning election microscopy results showed epithelial typical microvilli in both ADSCs and OMECs sheets.[Conclusions] ADSCs sheets can be used as new seed sources for ocular surface reconstruction.