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.

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
<ol>
	<li>Autoclave micro-dissection forceps and scissors (121 &#176;C, sterilization time: 20 min) and store them in a sterile container.</li>
	<li>Sterilize glass coverslips (24 mm x 24 mm) by incubating them in 1 M HCl for 24 hr on an orbital shaker at 37 &...

<ol>
	<li>Pagella, P., Jim&#233;nez-Rojo, L., Mitsiadis, T. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Roles+of+innervation+in+developing+and+regenerating+orofacial+tissues.">Roles of innervation in developing and regenerating orofacial tissues.</a> Cellular and molecular life sciences : CMLS. 71, 2241-2251 (2014).</li><li>Kumar, A., Brockes, J. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Nerve+dependence+in+tissue,+organ,+and+appendage+regeneration.">Nerve dependence in tissue, organ, and appendage regeneration.</a> Trends in neurosciences. 35 (11), 691-699 (2012).</li><li>Brownell, I., Guevara, E., Bai, C. B., Loomis, C. A., Joyner, A. 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The authors declare that they have no competing financial interests.

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...

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&#8211;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&#8211;11. Thus, it is important to analyse the yet neglected roles of innervation in the development of other important orofacial organs and tissues such as teeth.
In spite of the rich innervation of adult teeth, and in contrast to all other organs and tissues of the body, developing teeth start to be innervated at the earliest postnatal stages. Teeth develop as a result of sequential and reciprocal interactions between the oral ectoderm and cranial neural crest-derived mesenchyme. These interactions give rise to epithelial-derived ameloblasts and mesenchyme-derived odontoblasts that are responsible for the formation of enamel and dentin, respectively 12. Sensory nerves from the trigeminal ganglia and sympathetic nerves from the superior cervical ganglia innervate the adult teeth 13&#8211;15. During embryogenesis, nerve fibres emanating from the trigeminal ganglia project towards the developing tooth germs and progressively surround them but they do not penetrate into the dental papilla mesenchyme 13. Nerve fibres enter the dental pulp mesenchyme at more advanced developmental stages that correlate with odontoblast differentiation and dentin matrix deposition 16. Dental pulp innervation is completed soon after tooth eruption in the oral cavity 13. Previous studies have revealed that various semaphorins and neurotrophins are involved in the regulation of innervation during odontogenesis 16&#8211;19. Earlier studies have clearly demonstrated that innervation is a prerequisite for tooth formation in fishes 20. More recent studies have shown that homeostasis of dental mesenchyme stem cells in mouse incisors is regulated by sensory nerves via secretion of sonic hedgehog (shh) 21. Nevertheless, the role of innervation in tooth initiation, development and regeneration is still highly controversial in mammals 22&#8211;24.
A plethora of in vivo studies have provided important information about the patterns of innervation of dental tissues during development and repair processes of various animal models 13,25,26. 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 26&#8211;29. At the same time, co-culturing is subject to various technical adjustments. For example, nerves and specific dental tissues (e.g., dental pulp, dental follicle, dental epithelium) often require different culture media in order to guarantee tissue survival for long periods of time 30&#8211;32.
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 27&#8211;29. However, extension of the culture period is required to investigate the effects of innervation on tooth morphogenesis and cytodifferentiation, and to study the dynamics of nerve fibres branching within target organs. Therefore, non-contiguous co-cultures would be more suitable to perform studies on neuronal-dental tissues interactions.
Microfluidics systems allow co-cultures of neurons and different cell types in their appropriate culture media. In these devices, dental tissues and neurons are separated in different compartments, while allowing the growth of axons from the neural cell bodies through microchannels towards the compartment containing their target tissue 33. Microfluidic devices have been already used to study the interactions between neurons and microglia 34,35, as well as cell to cell interactions in cancer and neovascularization 35. Moreover, these systems have been used to study the interactions between dorsal root ganglia and osteoblasts 36.
We have recently demonstrated that trigeminal ganglia (TG) and teeth are able to survive for long periods of time when co-cultured in microfluidic devices 37. Moreover, we have demonstrated that teeth from different developmental stages maintain in these in vitro conditions the same repulsive or attractive effects on trigeminal innervation that they show in vivo 37. This protocol provides information about a simple, powerful 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.

<table border="0" cellpadding="0" cellspacing="0">
	<tbody>
		<tr>
			<td>AXIS Axon Isolation Devices</td>
			<td>Millipore</td>
			<td>AX15010-TC</td>
			<td>Microchannels of different lenght are available</td>
		</tr>
		<tr>
			<td>Laminin</td>
			<td>Sigma Aldrich</td>
			<td>L2020</td>
			<td></td>
		</tr>
		<tr>
			<td>Neurobasal</td>
			<td>Gibco</td>
			<td>21103-049</td>
			<td></td>
		</tr>
		<tr>
			<td>B27</td>
			<td>Gibco</td>
			<td>17504</td>
			<td></td>
		</tr>
		<tr>
			<td>Recombinant Mouse beta-NGF</td>
			<td>R&#38;D Systems</td>
			<td>1156-NG-100</td>
			<td>Human and Rat beta-NGF (R&#38;D Systems) are equivalent</td>
		</tr>
		<tr>
			<td>DMEM-F12</td>
			<td>Gibco</td>
			<td>11320-033</td>
			<td></td>
		</tr>
	</tbody>
</table>

analysis of developing tooth germ innervation using microfluidic co-culture devices

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.

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...

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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 ...

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8.2K Views.  University of Zurich. 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.