Development of Embryonic Mouse Heads Embedded in Alginate

Previously, we cultured 9.5 d embryonic mouse heads using an alginate encapsulation technique coupled with three dimensional culture in a rotation bioreactor. Cartilage growth in these heads was enhanced, perhaps due to lack of mass transfer in the bioreactor, and heads rounded up due to the rotational forces involved, so in the current study, heads encapsulated in alginate to preserve internal space and external morphology were cultured in a non-stick Petri dish. This allowed us to obviate hypoxia and rotational effects due to the bioreactor. Objective: The objective of this study was to assess development of embryonic mouse heads encapsulated in alginate and cultured in a Petri dish. Method: Embryos (E9) from ICR timed pregnant mice were decapitated between the second and third branchial arches, and heads were embedded in alginate and cultured in 100mm non-stick Petri dishes for 3 days. Samples were fixed on day 3 for photo-microscopic evaluation and future histology. Results: During the 3 days of culture, changes in shape of the head mirrored the progress in brain development: the prosencephalon enlarged anteriorly forming the telecephalon and the diencephalon. The telencephalon also developed lateral bulges, precursors of the cerebral hemispheres. The translucent roof of the myelencephalon was easily distinguished. Mesenchyme in the frontal process overgrew the nasal placode forming the nasal pits. The otic vesicle closed and separated from the epidermis, and the lens vesicle formed. Mandibular processes appeared fused, and no tongue was present. Differentiation of various tissues, including teeth, will be investigated in the histology portion using immunohistochemical staining. Conclusion: Alginate encapsulation is capable of supporting three-dimensional in vivo-like external development in encapsulated embryonic mouse heads and is an excellent system to study craniofacial problems. Supported by: NIH T32DE015355.