Analysis of Developing Tooth Germ Innervation Using Microfluidic Co-culture Devices

Co-cultures represent a valuable method to study the interactions between nerves and target tissues and organs. Microfluidic systems allow co-culturing ganglia and whole developing organs or tissues in different culture media, thus representing a valuable tool for the in vitro study of the crosstalk between neurons and their targets.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues. However, the mechanisms underlying these phenomena are not well understood yet. In particular, the role of innervation in tooth development and regeneration is neglected.

Several in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models. However, most of these approaches are not optimal to highlight the molecular basis of the interactions between nerve fibres and target organs and tissues.

Co-cultures constitute a valuable method to investigate and manipulate the interactions between nerve fibres and teeth in a controlled and isolated environment. In the last decades, conventional co-cultures using the same culture medium have been performed for very short periods (e.g., two days) to investigate the attractive or repulsive effects of developing oral and dental tissues on sensory nerve fibres. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation.

Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for a long period of time when co-cultured in microfluidic devices, and that they maintain in these conditions the same innervation pattern that they show in vivo.

On this basis, we describe how to isolate and co-culture developing trigeminal ganglia and tooth germs in a microfluidic co-culture system.This protocol describes a simple and flexible way to co-culture ganglia/nerves and target tissues and to study the roles of specific molecules on such interactions in a controlled and isolated environment.

Innervation plays a key role in the development, homeostasis and regeneration of organs and tissues ^1,2. Furthermore, innervation is involved in the regulation of stem cell proliferation, mobilization and differentiation ^3–5. Indeed, recent studies realised in tissues of the orofacial complex have shown that parasympathetic nerves are necessary for epithelial progenitor cells function during the development and regeneration of the salivary glands ^6,7. Similarly, it has been demonstrated that innervation is necessary for the development and maintenance of taste buds ^8–11....

All mice were maintained and handled according to the Swiss Animal Welfare Law and in compliance with the regulations of the Cantonal Veterinary office, Zurich.

1. Preparation of Dissection Material, Culture Media, Microfluidic Devices

1. Autoclave micro-dissection forceps and scissors (121 °C, sterilization time: 20 min) and store them in a sterile container.
2. Sterilize glass coverslips (24 mm x 24 mm) by incubating them in 1 M HCl for 24 hr on an orbital shaker at 37 &.......

These results show that isolated trigeminal ganglia can grow in one compartment of the microfluidic device and, in addition, that the development of the isolated tooth germs is sustained for a long period of time in the other compartment of the microfluidic device. Different culture media are used in the two compartments, and the microgrooves between the two compartments allow extension of axon from the trigeminal ganglion towards the developing tooth germs. Figure 3 represents a visualization of neurofi.......

Previous in vitro studies of tooth innervation were based on conventional co-cultures of trigeminal ganglia and dental tissues or cells ^26,28,29. These studies were conducted to investigate mainly the attractive effects of these cells or tissues on sensory axons ³⁸. Although bringing significant advances in the field, several technical issues were raised. Tooth germs start to degenerate after few days of culture ³⁷. Based on these observations, growing neurons and teeth in the sa.......

The work was funded by the University of Zurich. The authors would like to thank Estrela Neto and Dr. Meriem Lamghari for helping in establishing the co-culture conditions.