Efficient protocol for isolating human fibroblast from primary skin cell cultures: application to keloid, hypertrophic scar, and normal skin biopsies

Abstract

Abstract This protocol introduces a streamlined and efficient method for isolating human fibroblast from skin primary cell culture with a specific focus on its application to keloid, hypertrophic scar, and normal skin biopsies. Additionally, the absence of suitable animal models for keloid and hypertrophic scar has led preclinical research to rely on in vitro studies using primary cell cultures. This approach addresses the challenges of existing protocols in terms of time, cost, equipment, and technical expertise required. The method involves derivation, culture, and characterization analysis including cell proliferation, migration, and fibroblastic marker (Vimentin, CD90, CD73, and CD105) expression. Our study yielded high amounts of fibroblast from tested skin explants while maintaining their in vivo-like characteristics and behaviour. Immunostaining assay confirmed that the cultivated fibroblast was positively expressed Vimentin. Flowcytometry results showed high expression of CD90 and CD73 while relatively showing lower expression of CD105. Fibroblast derived from keloid tissue showed the highest rate of proliferation and migration ability compared to the other samples. These findings suggest an efficient and reproducible technique to cultivate high qualified fibroblast from human skin in normal or pathological condition, particularly for keloid and hypertrophic scar. The application of this protocol provides a foundation for further studies to investigate the progression and potential intervention of aberrant fibrotic dermatological disorder, in vitro.