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

Primary fibroblasts are widely used in a variety of experimental and therapeutic studies. Patient-derived skin biopsies are an accessible way to generate dermal fibroblasts for wound and burn therapeutics and can be easily reprogrammed to induced pluripotent stem cells (iPSCs). Despite the increasing use and interest in skin biopsies, there is limited information regarding the culturing potential of a single biopsy and the effects of extended culture on fibroblast formation and reprogramming potential. To better understand the potential of long-term skin biopsy culture, we cultured biopsy samples for 6-16 months, resulting in 6-16 generations of explant reculturing and then analyzed subsequent generations of fibroblasts. Our results showed that fibroblast morphology and physiology are maintained over time, but although older generations remained proliferative, they did so at a decreased rate. Gene expression analyses uncovered transcriptional changes with long-term skin culture, but deep DNA sequencing did not reveal any large deletions or amplifications. Spontaneous DNA mutations in fibroblast generations appeared to be random and not enriched for any specific signaling pathways. Importantly, fibroblasts generated after 16 months and over 16 generations in explant culture retained competency for reprogramming into induced pluripotent stem cells. Taken together, our results support long-term culture of skin biopsies to generate large numbers of primary fibroblasts. These cells maintain their identity and integrity, enabling the study of fibroblast maintenance as well as rare human disorders.