Mouse whole embryo culture: Evaluating the requirement for rat serum as culture medium

Abstract

Background Whole 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). Methods We 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. Results A 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. Conclusion Rat serum usage cannot be avoided, to achieve high quality mouse embryo cultures, but rat usage can be reduced using medium containing diluted serum.

Say NO to ROS: Their Roles in Embryonic Heart Development and Pathogenesis of Congenital Heart Defects in Maternal Diabetes

“…Additionally, culture conditions can be easily manipulated, for example using high glucose concentrations to mimic hyperglycemia. Although the rodent whole embryo culture was initially described about 60 years ago, because of these advantages, it is still being used to study organogenesis [167]. Chick embryo culture has also been used to study the effects of hyperglycemia on heart development.…”

Cannabidiol impairs neural tube closure in mouse whole embryo culture

“…Between E8.5 and E9.5, embryos develop prominent yolk sac circulation and undergo axial rotation (Figure 1a,b ). At the concentrations tested, CBD treatment does not significantly impact embryonic axial rotation (Figure 1c ), semi‐quantitively assessed using a previously described turning score (Culshaw, Savery, Greene, & Copp, 2019 ). Expansion of the yolk sac is also not impacted by CBD exposure (Figure 1d , only the 30 μM and associated vehicle groups were quantified).…”

Morphological phenotyping after mouse whole embryo culture

“…We culture mouse embryos by the rolling tube method ( New et al, 1973 ) using rat serum as culture medium, prepared as described ( Takahashi et al, 2014 ). Rat serum can be diluted up to 50:50 in certain culture media without loss of embryo quality ( Culshaw et al, 2019 ).…”

“…We focus particularly on phenotyping that can be performed at the bench, using only a stereomicroscope. Whole embryo culture (WEC) is a valuable tool in mammalian developmental biology ( New, 1978 ; Culshaw et al, 2019 ; Aguilera-Castrejon et al, 2021 ) that enables longitudinal analysis of developing mammalian embryos outside the uterine environment, thereby overcoming the limitations of viviparity for specific periods of embryogenesis. Table 1 lists some advantages and limitations of the WEC method, as regards availability and accessibility for study, the role of the maternal environment and aspects of experimental design.…”