Enhancing the Culture of Mouse Primary Fibroblasts to Study Age-Dependent Effects in Skin Tissue Engineering in Vitro

Introduction: Aged dermal fibroblasts exhibit reduced migration, proliferation, and extracellular matrix production, alongside upregulation of pro-inflammatory signaling that disrupts the proliferative, granulation, and remodeling phases of wound healing. Despite these age-related impairments, tissue engineering biomaterials are rarely evaluated using age-specific fibroblasts. Current in vitro skin aging models commonly rely on acute stressors, such as oxidative stress or DNA damage, which only partially recapitulate chronological aging. Alternatively, fibroblasts are artificially aged through extensive passaging, which may not reflect physiological aging. This work established a system for accurately representing chronological aging of mouse fibroblasts in vitro by isolating and evaluating fibroblasts from age-specific mice. While senescence in human fibroblasts results from telomere shortening, primary mouse fibroblasts senesce by oxidative damage in atmospheric O2, requiring careful culture conditions. Methods: We demonstrate that physiological O2 levels (3%) abrogate the detrimental effects of 20% O2 by comparing cell proliferation, senescence markers, and morphology of explant-derived fibroblasts. Next, fibroblasts from young (5 weeks), middle-aged (47 weeks), and old (90 weeks) mice were cultured (3% O2) and assessed for age-dependent changes in their proliferation, migration, immune signaling, collagen production, and metabolic activity in a 3D collagen matrix. Results: Our data demonstrate the impaired metabolic activity and migratory ability of old fibroblasts, as well as an overall amplification of pro-inflammatory proteins (e.g., MCP1 and IL-1α), which shifted tissue repair macrophages (“M2”) toward a pro-inflammatory (“M1”) phenotype. In 3D collagen-glycosaminoglycan scaffolds, old fibroblasts also showed significantly reduced metabolic activity compared to young and middle-aged cells. Conclusion: Together, these findings show that primary mouse fibroblasts can retain chronological age-related characteristics when cultured under physiological conditions in vitro, making them relevant in models of skin aging. Studying aging using these robust in vitro methods will be essential for improving the design and translational relevance of biomaterials and therapeutic strategies for wound healing and skin tissue engineering.