Comparative functional aspects of 3T3 fibroblasts in 2D and 3D cell-culture environment

Tissue engineering is a versatile and valuable approach that has been widely used for constructing tissue architecture and evaluating drug efficacy, among other applications. It particularly focuses on techniques aimed at restoring or replacing parts of or entire tissues and organs. The development of in-vitro bioengineered skin models holds a significant potential for clinical applications. Most existing methods for skin bioengineering primarily rely on two-dimensional (2D) cell cultures, which present notable limitations, particularly in assessing the safety and effectiveness of topical pharmaceutical agents due to the absence of dermal-epidermal interactions. In contrast, three-dimensional (3D) skin bioengineering models enable the culture of epidermal keratinocytes in combination with fibroblasts embedded within 3D matrices, better mimicking natural skin structure and function. In this study, we designed a comprehensive experimental approach to investigate the role of Swiss 3T3 fibroblast cells, and their variants pre-exposed to a single pulse of varying doses of Mitomycin C, in supporting the growth of keratinocytes when embedded in collagen. We examined the proliferation and viability of 3T3 cells to assess their ability to promote the growth of epidermal cells within this embedded niche. Additionally, we analysed the level of paracrine secretion of Keratinocyte Growth Factor (KGF) by fibroblasts in both conditions of 2D and 3D cell culture. Furthermore, we conducted a histological comparison of the epidermal layers and evaluated the expression of various epidermal markers to elucidate their structural and functional differences in 2D and 3D culture conditions. This study demonstrates the crucial role of both 2D and 3D skin bioengineering models and their potential applications in dermatological research and pharmaceutical development.