Comparative analysis of 3D and 2D cell-culturing methods in hair follicle spheroid morphogenesis and drug responsiveness.

Three-dimensional (3D) cell-culturing methods have usually been considered superior to two-dimensional (2D) culturing for in-vitro tissue formation intended for tissue engineering and drug research applications, including hair follicle (HF) development. However, cellular interactions within 3D cultures are generally more complex and therefore, may require further investigation. Apart from grafting in-vitro cultured (2D and 3D) dermal papilla cells directly onto the skin of animals to study the impact of 3D culturing on hair inductivity, molecular studies remain lacking in the understanding of how 2D and 3D culturing methods influence the morphogenesis of early stage HF models. The proposition that 3D cultures is always superior to 2D cultures for mimicking HF at its early developmental stage remains unknown. Therefore, this study aimed to investigate the influence of 3D and 2D culturing methods on the morphogenesis of HFs. 3D-cultured spheroids were assumed to exhibit greater expressions of HF-associated proteins and more expected drug-induced expression responses than 2D cultures. Dermal papilla cells and keratinocytes were cultured together in 2D and 3D cultures, where polyethylene glycol diacrylate microwell arrays were designed to provide the 3D culturing environment. Both 2D and 3D cultures were treated with either minoxidil or dihydrotestosterone (DHT) and the expressions of four hair proteins were analyzed. The results showed that 3D cultures responded in more expected ways than 2D cultures when exposed to minoxidil, demonstrating a significant increase in trichohyalin (AE15, one of the 4 proteins) as expected, while 2D cultures exhibited a significant down-regulation. On the other hand, surprisingly, DHT treatment significantly reduced all protein expressions in 2D culture as expected, but did not significantly alter protein expression in 3D culture, suggesting that 2D cultures could respond better than 3D cultures in DHT treatment.