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To avoid xenogeneic infection, we report a novel protocol for producing animal-derived component-free oral mucosal epithelial cells (OMECs) sheet for transplantation, in which collagenase was used to replace dispase II/trypsin-EDTA for digesting oral mucosal tissue, and human platelet-derived PLTMax to replace fetal bovine serum. The resulting epithelial aggregates were expanded on de-epithelialized amniotic membranes without 3T3 feeder cells, and serum-free EpiLife was used to reduce contamination by submucosal mesenchymal cells. The OMEC sheets thus generated showed similar positive keratin 3/76-positive and keratin 8-negative staining patterns compared with those generated by the original protocol. Colony formation efficiency assay, BrdU label retention assay, and p63 and p75 NTR  immunostaining results indicated that higher proliferative potentials and more progenitor cells were preserved by the modified protocol. TaqMan array analysis revealed that the transcription of integrin-linked kinase (ILK) was up-regulated along with an increase in β-catenin signaling and its downstream cell cycle modulators, cyclin D1 and p27 KIP1 . Furthermore, ILK silencing led to the inhibition of nuclear β-catenin accumulation, suppressed p63 expression, and reduced the expression of cyclin D1 and p27 KIP1 ; these observations suggest that ILK/β-catenin pathway may be involved in cell proliferation regulation during the  ex vivo  expansion of OMECs for transplantation purposes.

Preservation of epithelial progenitor cells from collagenase-digested oral mucosa during ex vivo cultivation