Abstract
BackgroundWhole embryo culture is a valuable research method in mammalian developmental biology and birth defects research, enabling longitudinal studies of explanted organogenesis‐stage rodent embryos. Rat serum is the primary culture medium, and can sustain growth and development over limited periods as in utero. However, the cost, labor, and time to produce culture serum are factors limiting the uptake of the methodology. The goal of replacing or at least reducing rat serum usage in culture would be in accordance with the principles of “replacement, reduction, and refinement” of animals in research (the 3Rs).MethodsWe performed cultures of mouse embryos for 24 hr from embryonic day 8.5 in serum‐free media or in rat serum diluted with defined media, compared with 100% rat serum. Developmental parameters scored after culture included yolk sac circulation, dorsal axial length, somite number, protein content, and completion of cranial neural tube closure.ResultsA literature review revealed use of both serum‐free and diluted rat serum‐based media in whole embryo culture studies, but with almost no formal comparisons of culture success against 100% rat serum. Two serum‐free media were tested, but neither could sustain development as in 100% rat serum. Dilution of rat serum 1:1 with Glasgow Minimum Essential Medium plus defined supplements supported growth and development as well as whole rat serum, whereas other diluent media yielded substandard outcomes.ConclusionRat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.
Highlights
Fate specification of neural plate border by canonical Wnt signaling and Grhl[3] is crucial for neural tube closure
The iron exporter ferroportin 1 is essential for development of the mouse embryo, forebrain patterning and neural tube closure
Growth of postimplantation mouse embryos cultured in a serum‐ supplemented, chemically defined medium
Summary
1. Miyazawa H., Yamamoto M., Yamaguchi Y., and Miura M. Mammalian embryos show metabolic plasticity toward the surrounding environment during neural tube closure. 2. Ivanovitch K., Temino S., and Torres M. Live imaging of heart tube development in mouse reveals alternating phases of cardiac differentiation and morphogenesis. 3. Huang Y., Wilkie R., and Wilson V. Methods for precisely localized transfer of cells or DNA into early postimplantation mouse embryos. A serum‐free and defined medium for the culture of mammalian postimplantation embryos. Comparisons of culture success (excluding effects of experimental treatments) No. K., Niehrs C., and Matsuo I. Fate specification of neural plate border by canonical Wnt signaling and Grhl is crucial for neural tube closure
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