Mouse embryonic fibroblast isolation, culture, and maintenance v1

This protocol outlines the step-by-step isolation, culture, and maintenance of mouse embryonic fibroblasts (MEFs) derived from CD1 strain embryos. MEFs are widely used as feeder layers to support the growth and self-renewal of a variety of stem cells in vitro (Bryja et al. 2006, Jain et al. 2014) including mouse trophoblast stem cells (Hayakawa et al. 2015). The protocol provides detailed instructions for embryo dissection, MEF culture and passaging, cryopreservation, thawing, and mitotic inactivation using mitomycin C. It includes guidance on cell confluency and density to optimise viability and expansion, as well as medium recipes, reagent reference tables, and safety notes to support reproducibility and ease of use for both new and experienced researchers. MEFs are isolated at embryonic day (E)13.5 by carefully removing extraembryonic tissues, followed by mechanical and enzymatic dissociation of the embryonic body. To function effectively as feeder cells, MEFs are rendered mitotically inactive to prevent overgrowth while retaining the metabolic activity necessary to support stem cell cultures. This is typically achieved through γ-irradiation or chemical treatment with mitomycin C (Nagy et al 2006). While γ-irradiation requires specialised equipment, mitomycin C offers a more accessible alternative, although it must be handled with care due to its cytotoxic nature. Thorough washing is essential after inactivation to avoid residual toxicity that could affect downstream applications. By standardising each step of MEF preparation and maintenance, this protocol ensures consistent cell quality and optimised experimental performance. It serves as a comprehensive resource for researchers working in stem cell biology, developmental biology, and cell culture-based applications.