Abstract
In our previous study, we found that dry-preserved multilayered fibroblast cell sheets promoted angiogenesis and wound healing in a mouse ulcer model by releasing high levels of intracellular fibroblast growth factor 2 (FGF2), hepatocyte growth factor (HGF), and vascular endothelial growth factor (VEGF), from dried cells. In the present study, to identify which cell types are suitable for human dry-preserved cell sheets (dry sheets), we compared the intracellular FGF2 levels in seven types of cells reported as cell sheets for clinical use or preclinical studies. FGF2 levels were high in mesenchymal cells, including human oral fibroblasts (HOFs) and human dermal fibroblasts (HDFs), human dental pulp stem cells (DPSCs), and human mesenchymal stem cells (MSCs); in contrast, FGF2 levels in human umbilical vascular endothelial cells (HUVECs), human skeletal muscle myoblasts (SkMMs), and human epidermal keratinocytes (HEKs) were remarkably low, approximately 25% those in fibroblasts. In addition, we prepared dry sheets from HOFs, DPSCs, and MSCs, and analyzed the growth factors released from each dry sheet upon rehydration. High levels of FGF2, HGF, and VEGF were detected in the eluate prepared by immersing each dry sheet. In particular, FGF2 and HGF were the most abundant in HOFs. An in vitro cell proliferation assay showed that these eluates significantly enhanced HUVEC proliferation compared to control cells. Furthermore, cells incubated with HOF eluate showed significantly higher cell proliferation than cells incubated with DPSC and MSC eluates. However, this proliferative response was significantly blocked by FGF2-neutralizing antibodies. These results demonstrate that growth factors released from human dry sheets have physiological activity and that this activity is mainly mediated by the effect of FGF2. Fibroblasts are ideal for the clinical application of dry-preserved cell sheets in humans owing to their high intracellular FGF2 content, fast cell proliferation, ease of handling, availability, and low culture costs, making them the most suitable cell source for regenerative medicine, with FGF2 release as the mechanism of action.