In recent years, a variety of techniques have been developed to engineer tissues composed only of cells and the matrix materials that they secrete without any exogenous scaffold materials. Such techniques are even being applied for tissues where scaffolds have shown success (e.g., skin, bone, and cartilage) [18]. Scaffold-free cell sheet-based constructs have been applied for tissue repair due to their high rates of nutrient diffusion, abundant deposition of ECM, and interactions with cell membrane proteins, including growth factors, ion channels, and cell-to-cell junction proteins. Ascorbic acid has been reported to induce telomerase activity, leading to upregulation of type I collagen, fibronectin, and integrin β1. Ascorbic acid-treated mesenchymal cells form sheets due to increased ECM production. Furthermore, Lee et al. constructed a dermal equivalent from fibroblasts treated with ascorbic acid without other exogenous materials [19]. Based on this paper, we cocultured 3 types of cells (keratinocytes, dermal fibroblasts, and dermal microvascular endothelial cells) to form scaffold-free skin equivalents containing capillary-like networks. The cocultured cells expressed high levels of vascularization-associated growth factors, including VEGF, bFGF, and PDGF, compared to cocultures of fibroblasts and endothelial cells and monocultures.
