Biological and mechanical influence of three-dimensional microenvironment formed in microwell on multicellular spheroids composed of heterogeneous hair follicle stem cells

Abstract

Hair loss caused by malfunction of the hair follicle stem cells (HFSCs) and physical damage to the skin is difficult to recover from naturally. To overcome these obstacles to hair follicle (HF) regeneration, it is essential to understand the three-dimensional (3D) microenvironment and interactions of various cells within the HFs. Therefore, 3D cell culture technology has been used in HF regeneration research; specifically, multicellular spheroids have been generally adapted to mimic the 3D volumetric structure of the HF. In this study, we culture HF-derived cells, which are mainly composed of HFSCs, in the form of 3D spheroids using a microwell array and discuss the effects of the 3D cellular environment on HF morphogenesis by expression measurements of Sonic hedgehog signaling and stem cell markers in the HF spheroids. Additionally, the influences of microwell depth on HF spheroid formation and biological conditions were investigated. The biomolecular diffusion and convective flow in the microwell were predicted using computational fluid dynamics, which allows analysis of the physical stimulations occurring on the spheroid at the micro-scale. Although a simple experimental method using the microwell array was adopted in this study, the results provide fundamental insights into the physiological phenomena of HFs in the 3D microenvironment, and the numerical analysis is expected to shed light on the investigation of the geometric parameters of the microwell system.