Name: Analiza rozwoju unerwienia zarodków zębów za pomocą urządzeń do kohodowli mikroprzepływowej (Wideo) | JoVE Journal
Uploaded: 2015-08-14T14:00:00.000+00:00
Duration: 8 min 1 s
Description: Poznaj metodę izolacji i współkultury zarodkowych ganglionów nerwu trójdzielnego oraz zawiązków zębów myszy przy użyciu mikroprzepływowych urządzeń. Technika ta umożliwia badanie interakcji nerwowych z tkankami docelowymi w kontrolowanych warunkach in vitro.

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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 &#176;C. Wash them three times with sterile, distilled H2O and three times with ethanol 99%. Dry then the coverslips at 37 &#176;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%.</li>
	<li>Remove carefully the AXIS Axon Isolation Devices from the package using sterile forceps and place them in a sterile Petri dish.</li>
	<li>Using a sterile biopsy punch (&#248;: 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.</li>
	<li>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.</li>
	<li>Place each coverslip into a 35 mm Petri dish or into a well within a 6-wells plate.</li>
	<li>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.</li>
	<li>In each culture chamber, pipette 150 &#181;l 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.</li>
	<li>If air can still be seen within the chambers, re-pipette the poly-D-lysine solution into the chambers.</li>
	<li>Incubate the devices with poly-D-lysine O/N&#160;at 37 &#176;C.</li>
	<li>Wash chambers three time with sterile, distilled H2O.</li>
	<li>Fill chambers with 150 &#181;l laminin working solution (Sigma-Aldrich, 5 &#181;g/ml, in PBS or serum-free medium), and incubate O/N at 37 &#176;C.</li>
	<li>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.</li>
	<li>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 &#181;g/ml ascorbic acid.</li>
</ol>
2. Mouse Embryo Generation and Dissection
<ol>
	<li>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.</li>
	<li>Clean the dissection area and the stereoscope with ethanol 70%.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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</li>
	<li>Dissect out the embryos from the uterus and free them from their extraembryonic tissues. Place the embryos in PBS on ice.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
	<li>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.</li>
</ol>
3. Microfluidic Co-cultures
<ol>
	<li>After dissection, remove laminin from the microfluidic devices. Fill the chambers with 200 &#181;l of the respective media.</li>
	<li>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.</li>
	<li>Culture the samples in incubator at 37 &#176;C, 5% CO2.</li>
	<li>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.</li>
	<li>During the culture period, co-cultures can be easily imaged by time-lapse microscopy. Co-cultures can be maintained for over 10 days.</li>
	<li>After the culture period, wash the chambers by pipetting 150 &#181;l of PBS into one well per chamber, and letting PBS flow through the chambers three times.</li>
	<li>Remove the PBS and fix the samples by pipetting 150 &#181;l of paraformaldehyde 4% (in PBS) in one well per chamber; incubate at RT for 15 min.</li>
	<li>Wash the chambers twice with PBS as described in 3.6.</li>
	<li>Proceed with further analysis.</li>
</ol>

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</ol>

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><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&amp;amp;D Systems</td><td>1156-NG-100</td><td>Human and Rat beta-NGF (R&amp;amp;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

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...

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 ...

The overall goal of this procedure is to show how to isolate and co-culture the developing trigeminal ganglia and tooth germs. This is accomplished by first sterilizing, mounting and coating the microfluidic devices. The second step is to dissect trigeminal ganglia and tooth germs from a specific developmental stage in the mouse of interest.Next, the trigeminal ganglia and the tooth germs are placed into the microfluidic devices and co cultured. The final step is to fix the cells and stain them using immunofluorescence. Ultimately, microscopy is used to show the innervation pattern that is the result of co-culture.The main advantage of this technique of existing methods, such as conventional co cultures, is that this method allows the co culture of different organs and tissues each in their own optimal culture.Medium. This method can help answer key questions in the field of artificial innovation, such as the role of different molecules on tooth innovation and the rule of innovation in tooth development. Begin by autoclaving dissection tools, which include a pair of microdissection forceps and a pair of scissors.Next, sterilize a batch of 24 millimeter by 24 millimeter glass cover

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Research

JoVE Journal

Neuroscience

This protocol describes a microfluidic co-culture model for simultaneous and localized culture of epithelial cells and bacteria. This model can be used for investigating the role of different soluble molecular signals on pathogenesis as well as screen the effectiveness of putative probiotic bacterial strains.

Microfluidic Co-culture of Epithelial Cells and Bacteria for Investigating Soluble Signal-mediated Interactions

This video demonstrates the utilization of a microfluidic device to study the interactions between tooth germ pulp and trigeminal ganglia. It showcases the placement of tissues inside the microfluidic device and provides a detailed visualization of trigeminal neurite growth toward the tooth germ through the device's microgrooves. Additionally, it highlights the role of tooth-derived molecular factors in promoting or inhibiting neurite extension into the tooth germ pulp, which is crucial for...

Analyzing Tooth Germ and Trigeminal Ganglia Interactions Using a Microfluidic Co-culture System

Microfluidic oxygen control confers more than just convenience and speed over hypoxic chambers for biological experiments. Especially when implemented via diffusion through a membrane, microfluidic oxygen can provide simultaneous liquid and gas phase modulations at the microscale-level. This technique enables dynamic multi-parametric experiments critical for studying islet pathophysiology.

Quantitative and Temporal Control of Oxygen Microenvironment at the Single Islet Level

Here we detail a method to culture tooth germs in mandible slices using a tissue chopper. This method allows unique access to the tooth during development, providing excellent opportunity for manipulation and lineage tracing, not available using more traditional culture methods.

The Slice Culture Method for Following Development of Tooth Germs In Explant Culture

We describe the isolation, dispersion and plating of dental pulp (DP) primary cells with trigeminal (TG) neurons cultured atop overlying transwell filters. Cellular responses of DP cells can be analyzed with immunofluorescence or RNA/protein analysis. Immunofluorescence of neuronal markers with confocal microscopy permits the analysis of neurite outgrowth responses.

A Co-Culture Method to Study Neurite Outgrowth in Response to Dental Pulp Paracrine Signals

The Trowell-type organ culture method has been used to unravel complex signaling networks that govern tooth development and, more recently, for studying regulation involved in stem cells of the continuously growing mouse incisor. Fluorescent-reporter animal models and live-imaging methods facilitate in-depth analyses of dental stem cells and their specific niche microenvironment.

Use of Trowell-Type Organ Culture to Study Regulation of Dental Stem Cells

This protocol presents a method for the culture and 3D growth of ameloblast-like cells in microgravity to maintain their elongated and polarized shape as well as enamel-specific protein expression. Culture conditions for the culture of periodontal engineering constructs and lung organs in microgravity are also described.

Propagation of Dental and Respiratory Cells and Organs in Microgravity

We present a protocol to develop epithelial organoid cultures starting from human tooth. The organoids are robustly expandable and recapitulate the tooth's epithelial stem cells, including their ameloblast differentiation capacity. The unique organoid model provides a promising tool to study human dental (stem cell) biology with perspectives for tooth-regenerative approaches.

Establishing Organoids from Human Tooth as a Powerful Tool Toward Mechanistic Research and Regenerative Therapy

neuroHuMiX is an advanced gut-on-a-chip model to study the interactions of bacterial, epithelial, and neuronal cells under proximal and representative co-culture conditions. This model allows unravelling of the molecular mechanisms underlying the communication between the gut microbiome and the nervous system.

A Gut-on-a-Chip Model to Study the Gut Microbiome-Nervous System Axis

Ex vivo live imaging is a powerful technique for studying the dynamic processes of cellular movements and interactions in living tissues. Here, we present a protocol that implements two-photon microscopy to live track dental epithelial cells in cultured whole adult mouse incisors.

Using Ex Vivo Live Imaging to Investigate Cell Divisions and Movements During Mouse Dental Renewal

Poznaj metodę izolacji i współkultury zarodkowych ganglionów nerwu trójdzielnego oraz zawiązków zębów myszy przy użyciu mikroprzepływowych urządzeń. Technika ta umożliwia badanie interakcji nerwowych z tkankami docelowymi w kontrolowanych warunkach in vitro.

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

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