Long-term culture of skin biopsies: Maintenance of fibroblast production and competency of reprogramming

Primary fibroblasts are a commonly used cell type used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate fibroblasts for use in various assays as well as reprogramming to iPSCs. To understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6 to 16 months and analyzed subsequent generation of fibroblasts. We found maintenance of morphology and physiology over time. Proliferation assays showed that older generations remained proliferative but at a decreased rate. mRNA analyses revealed transcriptional changes with long-term skin culture. Deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations seemed to be random and not enriched for any specific signaling pathways. Older fibroblasts generated after 16 months in culture retained competency for reprogramming into iPSCs. Our results support long term culture of skin to generate very large numbers of primary fibroblasts. These cells maintain their identity and integrity enabling the study of human disorders, particularly rare ones. To determine the effect of long-term skin biopsy culture and the stability of produced fibroblasts, we cultured 2 skin biopsies for 6 to 16 generations We extracted RNA from fibroblasts from Gen1, Gen6, and Gen16 and compared expression changes over time to determine culture stability