Isolation, Validation, and Long-Term Culture of Mouse Ear Fibroblasts

In contrast, minimal media (DMEM + 10% FBS) proved sufficient for both isolation and long-term culture, maintaining fibroblast identity through at least 13 passages. Cells displayed characteristic fibroblast morphology, expressed the mesenchymal marker vimentin, and maintained fibroblast-associated transcripts with minimal epithelial and adipocyte marker expression throughout culture. Bulk RNA sequencing revealed 17% of mapped genes exhibited differential abundance between passage 3 and passage 9, with enrichment in functional categories related to cell cycle regulation and paracrine signaling.

This protocol provides an accessible primary fibroblast cell culture model with broad research applications emphasizing reproducibility and resource- conscious practices.