We have performed clinical translation of epithelial cell sheets fabricated on temperature-responsive culture surfaces to treat cornea and esophagus. In the preclinical study using animal models, we found epithelial cell growth potential varied among species. Canine epithelial cell growth was prominent, while rat one was poor under 3T3 feeder layer-free condition. The aim of the present study was to identify growth-promoting factors for epithelial cells. Conditioned medium of canine cell culture harvested at different time points showed different growth promotive activity for rat epithelial cells. Time-dependent gene expression was quantitatively evaluated for forty growth factors, and compared with conditioned medium results. Statistically significant promotive activity was observed with IL-1RA, and significant inhibitory activity was observed with IL-1. Furthermore, neutralizing anti-IL-1 antibody also showed significant promotive activity. Human epidermal keratinocytes were promoted to proliferate by IL-1RA and neutralizing anti-IL-1 antibody, and showed well differentiation to form transplantable, squamous stratified epithelial cell sheets. These findings would be useful to fabricate reproducible, transplantable epithelial cell sheets for regenerative medicine.