Mouse Embryonic Tooth Germ Dissection and Ex vivo Culture Protocol

Background

Teeth are considered to represent a typical epithelial-mesenchymal organ. When investigating tooth development, observation of the developmental pattern can help to elucidate the mechanisms of morphogenesis biology. When transfecting siRNAs or expression vectors into embryonic organs, a maternal injection protocol is commonly used in studies of mice. However, with traditional methods, it is difficult to efficiently transport reagents into the target organ. On the other hand, an ex vivo organ culture simplifies direct transportation of reagents into a dissected organ, allowing for observations in a three-dimensional manner. In addition to teeth, other epithelial-mesenchymal organs such as salivary gland and hair can also be successfully cultured with this system.

Several different organ culture protocols have been presented for tooth germ organ culturing, such as a semi-solid method (Heywood and Appleton, 1984), with Matrigel (Shih and Sander, 2014; Sun et al., 2016), and with the use of mesh placed in cell culture dishes (Yamada et al., 1980; Bringas et al., 1987; Tabata et al., 1996; Fukumoto et al., 2006; Yamamoto et al., 2008; Yamada et al., 2016). However, each requires preparation of the culture materials in the wells every time prior to organ dissection. In contrast, with the present organ culture system, the materials can be directly purchased from a supplier, which saves time and avoids inevitable contamination during preparation of the culture materials. Traditional methods used to examine the functions of gene or drugs in vivo require time to determine the results individually, whereas with an ex vivo culture, it is possible to examine a large number of organ samples at the same time. Furthermore, it is possible to screen for critical genes or other related factors during organ development.

Here, we describe a relatively simple and efficient method for ex vivo organ culture of embryonic tooth germs. With the use of this protocol, screening studies and development observations can be objectively and directly conducted.