Isolation, validation, and long-term culture of mouse ear fibroblasts.

Primary fibroblasts are essential cell culture models for studying cellular homeostasis, disease mechanisms, and host-pathogen interactions. Primary cells offer physiological relevance that immortalized cell lines cannot recapitulate. However, existing protocols for isolating and culturing primary fibroblasts lack standardization for long-term applications. Contamination by non-fibroblast cell types, variable media formulations, and poorly monitored passage numbers remain critical challenges for reproducibility. Here, we present a streamlined, cost-optimized protocol for isolating and culturing mouse ear fibroblasts (MEarFs) suitable for extended passages. We demonstrate that enriched media (Advanced DMEM/F-12+15% FBS) consistently promoted adipocyte-like cell contamination in long-term MEarF cultures. In contrast, minimal media (DMEM+10% FBS) proved sufficient for both isolation and long-term cultures, with fibroblast gene expression confirmed through passage 9. Cells displayed characteristic fibroblast morphology, expressed the mesenchymal marker vimentin, and maintained fibroblast-associated transcripts with minimal epithelial and adipocyte marker expression throughout the culture. Bulk RNA sequencing revealed that 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.