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

Summary

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.

Abstract

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.

Introduction

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. 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 ^13–15. 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 ^16–19. 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 ^22–24.

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 ^26–29. 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 ^30–32.

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 ^27–29. 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.

Protocol

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 °C. Wash them three times with sterile, distilled H₂O and three times with ethanol 99%. Dry then the coverslips at 37 °C or under sterile flow hood. Finally, autoclave or expose the coverslips to UV light (30 min) to complete the sterilization. Coverslips can then be stored in ethanol 70%.
3. Remove carefully the AXIS Axon Isolation Devices from the package using sterile forceps and place them in a sterile Petri dish.
4. Using a sterile biopsy punch (ø: 1mm) create one hole per sample to be cultured (Figure 1) in correspondence of the culture chambers.
   NOTE: Do not punch too close to the microgrooves, as they might be damaged by the pressure applied.
5. Sterilize the AXIS Axon Isolation Devices by immerging them in ethanol 70%. Dry then AXIS Axon Isolation Devices and coverslips completely under a sterile flow hood. Wait a minimum of 3 hr before proceeding.
   NOTE: incomplete drying will result in defective assembly of the microfluidic devices.
6. Place each coverslip into a 35 mm Petri dish or into a well within a 6-wells plate.
7. Place the AXIS Axon Isolation Device onto the coverslip and press gently but firmly with a forceps with bent ends in order to allow full adhesion between the isolation device and the glass coverslip.
8. In each culture chamber, pipette 150 µl of poly-D-lysine (0.1 mg/ml in sterile, distilled H₂O). Place the microfluidic devices under vacuum for 5 min, in order to remove all the air from the culture chambers.
9. If air can still be seen within the chambers, re-pipette the poly-D-lysine solution into the chambers.
10. Incubate the devices with poly-D-lysine O/N at 37 °C.
11. Wash chambers three time with sterile, distilled H₂O.
12. Fill chambers with 150 µl laminin working solution (Sigma-Aldrich, 5 µg/ml, in PBS or serum-free medium), and incubate O/N at 37 °C.
13. Prepare 50 ml of medium for trigeminal ganglia cultures ³⁷ , composed as follows: 48 ml Neurobasal medium, 1 ml B-27, 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 5 ng/ml nerve growth factor (NGF), 0.25 pM cytosine arabinoside.
14. Prepare 50 ml of medium for tooth germ cultures ³⁷, composed as follows: 40 ml DMEM-F12, 10 ml foetal bovine serum (FBS, final concentration: 20%), 100 U/ml penicillin/streptomycin, 2 mM L-glutamine, 150 µg/ml ascorbic acid.

2. Mouse Embryo Generation and Dissection

1. Determine embryonic age according to vaginal plug (vaginal plug: embryonic day of development 0.5, E0.5) and confirm it via morphological criteria. For this protocol, we generally use E14.5-E17.5 mouse embryos.
2. Clean the dissection area and the stereoscope with ethanol 70%.
3. Sacrifice the pregnant mother via cervical dislocation. Block the neck of the mouse with the first and second finger onto a grid, and pull with decision the tail.
4. Dissect the skin around the lower abdomen, and open the abdomen using scissors. Locate the uterus: during such late stages of pregnancy, the uterus abundantly fill the abdominal cavity.
5. Dissect out the uterus and place in a tube filled with PBS on ice. When on ice, the tissue can be left for several hr. Discard the corpse of the mother according to institutional guidelines
6. Dissect out the embryos from the uterus and free them from their extraembryonic tissues. Place the embryos in PBS on ice.
7. Decapitate the embryos using scissors, and separate the lower jaw from the rest of the head using micro-dissection scissors (Figure 2A). Remove precisely the lower jaw, without damaging the trigeminal ganglia; as the latter are localized in close proximity to the lower jaw, their accidental damage is possible. Preserve the lower jaw and the rest of the head in cold PBS, on ice.
8. To dissect TG, take the head and place it onto a dissection glass Petri dish, previously filled with cold PBS. Using the forceps, remove the skin and the skull. Remove then the telencephalon and the cerebellum by placing forceps below the telencephalon and lift; the telencephalon and the cerebellum will flip together, leaving the bottom of the skull exposed.
9. Localize the trigeminal ganglia (shown in Figure 2B). Use the forceps to separate the TG from the trigeminal nerves. Eliminate the remnants of the trigeminal projections using the dissection needles as knifes. Place the dissected TG in a Petri dish filled with cold PBS and keep them on ice.
10. To dissect embryonic teeth, place the lower jaw, previously separated from the skull, onto the dissection glass Petri dish, filled with cold PBS. Using dissection needles as knifes, remove the tongue and the skin surrounding the jaw. Separate the left and the right hemi-jaws by cutting along the midline of the jaw. The tooth germs are easily visible, as shown in Figure 1C. Isolate the tooth germs using dissection needles and remove the excess of non-dental tissues. Place the dissected tooth germs in a Petri dish filled with cold PBS and keep them on ice.

3. Microfluidic Co-cultures

1. After dissection, remove laminin from the microfluidic devices. Fill the chambers with 200 µl of the respective media.
2. With forceps, transfer gently the dissected TG and tooth germs into the holes created by punching (Figure 1D). Make sure that the tooth germs do not float and that they sink until they contact the coverslips.
3. Culture the samples in incubator at 37 °C, 5% CO_2.
4. Change the culture medium every 48 hr. Do not empty the chambers completely, and do not pipette directly into the culture chambers. Complete emptying of chambers would result in the formation of air bubbles within the chambers; direct pipetting into the chamber would result in axonal damage. To avoid these issues, remove the medium pointing the pipette towards the external side of the wells; similarly, pipette the fresh medium on the side of the wells located opposite to the chambers.
5. During the culture period, co-cultures can be easily imaged by time-lapse microscopy. Co-cultures can be maintained for over 10 days.
6. After the culture period, wash the chambers by pipetting 150 µl of PBS into one well per chamber, and letting PBS flow through the chambers three times.
7. Remove the PBS and fix the samples by pipetting 150 µl of paraformaldehyde 4% (in PBS) in one well per chamber; incubate at RT for 15 min.
8. Wash the chambers twice with PBS as described in 3.6.
9. Proceed with further analysis.

Results

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...

Discussion

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...

Materials

Name	Company	Catalog Number	Comments
AXIS Axon Isolation Devices	Millipore	AX15010-TC	Microchannels of different lenght are available
Laminin	Sigma Aldrich	L2020
Neurobasal	Gibco	21103-049
B27	Gibco	17504
Recombinant Mouse beta-NGF	R&D Systems	1156-NG-100	Human and Rat beta-NGF (R&D Systems) are equivalent
DMEM-F12	Gibco	11320-033