Sarah A. Alfaqeeh is funded by Kind Saud University College of Dentistry, Ministry of Higher Education, Kingdom of Saudi Arabia.

1. Set Up
<ol>
	<li>Sharpen the dissection instruments (using a sharpening stone lubricated with mineral oil) and sterilize prior to use for organ culture using a 70% ethanol spray and dry-heat sterilization. Sharpen the dissection needles using electrolysis in 2 M sodium hydroxide using a 12 V supply.</li>
	<li>Prepare the culture medium used for the dissection and culture of embryonic organs, consisting of Advanced Dulbecco&#39;s Modified Eagle Medium F12 (DMEM F12) supplemented with 1% GlutaMAX&#160;and 1% penicillin-streptomycin.</li>
</ol>
2. Embryo Dissection
<ol>
	<li>Cull a timed mated pregnant female using a schedule one method as approved by the Home Office or other governing body. Dissect away the skin around the lower abdomen, and cut open the abdomen using scissors. Locate the two uterine horns, which connect together at the lower midline.</li>
	<li>Dissect out the uterus and place in a tube filled with medium on ice. When on ice, the tissue can be left for several hours.</li>
	<li>Remove the uterus and place in a Petri dish in medium under the microscope. Where possible embryo dissections should be performed in a laminar-flow hood to minimize contamination.</li>
	<li>Dissect out the embryos from the uterus and free them from their extraembryonic tissues.</li>
	<li>Decapitate the embryos using a tungsten needle and transfer the heads into a fresh dish of medium.</li>
	<li>Isolate the mandibles from the heads by placing a needle into the oral cavity and cut through towards the back of the head (Figures 1A and&#160;1B). Isolated mandibles can be temporarily stored in medium on ice while waiting for chopping.</li>
	<li>For whole mandible slices stages E11.5-E15.5 are ideal for tooth germ culture. After E15.5 the developing bone in the mandible is too hard for chopping. Tooth slices can still be made but the bone (dentary and forming alveolar bone) must first be removed by dissection before chopping9. Remove bone carefully using tungsten needles and forceps.</li>
</ol>
3. Embryo Slicing
<ol>
	<li>Prepare the mandible tissue slices using a standard table tissue chopper (Figure 1C). Use a fresh blade after approximately 200 mandibles, and make sure it lies flush with the mounting disc when it falls.</li>
	<li>Clean the mounting disc and blade with 70% ethanol before use. Care must be taken when using the machine, as the blade is very sharp.</li>
	<li>Set the chopper at a cutting distance of between 200-400 &#956;m according to the age of the specimen, and depending on whether the whole tooth germ is required. Set the blade force on maximum.</li>
	<li>Transfer a mandible to the plastic mounting disc using a pipette or forceps. Arrange the mandible on the disc making sure the orientation is still clear. The developing tongue can be used as a useful landmark for determining orientation. For molars use a frontal/transverse orientation (see plane of chopping in Figure 1B), while for incisors use a sagittal slice through the mandible to reflect the different orientation of these teeth as they grow (see plane of chopping in Figure 2A).</li>
	<li>Remove the excess medium from around the tissue using filter paper in order to slightly dry the mandible on the mounting disc.</li>
	<li>Immediately after drying, place the disc on the circular stainless steel table of the chopper. Turn the machine on and press start. The table traverses automatically from left to right while at the same time the chopping arm carrying the blade is raised and dropped at up to 200 strokes/min.</li>
	<li>Turn off the machine once the tissue has been completely sliced. Make sure the blade arm is in the raised position and remove the disc. Do not place fingers under the blade arm or leave the machine unattended when chopping.</li>
	<li>Take the mounting disc and immediately add medium to the slices on the disc in order to prevent excessive drying. Carefully dislodge the sliced tissue from the bottom of the disc using a tungsten needle and then pipette into a small Petri dish of medium (Figure 2A).</li>
	<li>Separate the slices using a needle and then select the tissue slices containing the tissue of interest under a stereomicroscope (Figures 1D, 1E,&#160;and 2B). Place selected slices in fresh culture dishes with medium.</li>
</ol>
4. Slice Culture
<ol>
	<li>Prepare central-well organ culture dishes ready for culture by adding approximately 2 ml of autoclaved water to the outer well to prevent dehydration, and by positioning a metal grid across the center of the dish (Figures 3A and 3B).</li>
	<li>Prepare metal grids by cutting strips approximately 4 cm x 1.5 cm&#160;from sheets of stainless steel mesh. Use a hole punch to make a hole in the center and bend the sides to create a staggered end. The grid should fit flat across the central well, lying parallel to the bottom of the dish (Figures 3A and 3B). Autoclave the grids to sterilize before use.</li>
	<li>Cut a piece of transparent polyethylene terephthalate (PET) membrane (pore size 0.4 &#956;m) large enough to cover the hole in the grid. Place the cut membrane into the dish with the selected slice.</li>
	<li>Place the slice on top of the membrane and then carefully lift the membrane out of the medium, with slice flattened in the center.</li>
	<li>Place the filter on the grid over the circular hole in the center, and add approximately 1 ml of culture medium to the well, up to the level of the membrane.</li>
	<li>For manipulations add proteins or small molecule inhibitors to the medium (for example8).</li>
	<li>Photograph the slice for a record of morphology at day 0 of culture.</li>
	<li>Place the dishes in a humidified atmosphere of 5% CO2 at 37 &#176;C in an incubator.</li>
	<li>Photograph the cultures at regular intervals for on average 7 days. Change the culture medium every 48-72 hr. For photography view the slices with a light source from underneath (Figures 2C-E).</li>
</ol>
5. Lineage Tracing
If lineage tracing is required, lipophilic dyes such as DiI or DiO can be injected into the tooth germ before step 4, slice culture.
<ol>
	<li>Pull glass capillary needles using a needle puller.</li>
	<li>Break the tip of the needle using forceps and place tip down into the dye solution. Leave for a few minutes while the dye fills the tip by capillary action.</li>
	<li>Place the filled needle into a holder and attach via tubing to an injector or mouth aspirator.</li>
	<li>Position the tip of the needle in the culture medium touching the area of the slice due to be labeled. Apply air pressure to displace a small amount of dye out of the needle and onto the tissue.</li>
	<li>Remove needle and check for labeling under fluorescence.</li>
	<li>Successfully labeled slices can be transferred to filters and cultured as described in step 4.</li>
</ol>

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	<li>Rothova, M., Peterkova, R., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Fate+map+of+the+dental+mesenchyme:+dynamic+development+of+the+dental+papilla+and+follicle.">Fate map of the dental mesenchyme: dynamic development of the dental papilla and follicle.</a> Developmental biology. 366, 244-254 (2012).</li><li>Ferguson, C. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Activin+is+an+essential+early+mesenchymal+signal+in+tooth+development+that+is+required+for+patterning+of+the+murine+dentition.">Activin is an essential early mesenchymal signal in tooth development that is required for patterning of the murine dentition.</a> Genes &amp; development. 12, 2636-2649 (1998).</li><li>Trowell, O. A. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+culture+of+mature+organs+in+a+synthetic+medium.">The culture of mature organs in a synthetic medium.</a> Experimental cell research. 16, 118-147 (1959).</li><li>Matalova, E., Antonarakis, G. S., Sharpe, P. T., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+lineage+of+primary+and+secondary+enamel+knots.">Cell lineage of primary and secondary enamel knots.</a> Developmental dynamics : an official publication of the American Association of Anatomists. 233, 754-759 (2005).</li><li>Mitsiadis, T. A., Tucker, A. S., De Bari, C., Cobourne, M. T., Rice, D. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=A+regulatory+relationship+between+Tbx1+and+FGF+signaling+during+tooth+morphogenesis+and+ameloblast+lineage+determination.">A regulatory relationship between Tbx1 and FGF signaling during tooth morphogenesis and ameloblast lineage determination.</a> Developmental biology. 320, 39-48 (2008).</li><li>Diep, L., Matalova, E., Mitsiadis, T. A., Tucker, A. S. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Contribution+of+the+tooth+bud+mesenchyme+to+alveolar+bone.">Contribution of the tooth bud mesenchyme to alveolar bone.</a> Journal of experimental zoology. Part B, Molecular. 312B, 510-517 (2009).</li><li>Rothova, M., Feng, J., Sharpe, P. T., Peterkova, R., Tucker, A. 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W., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+primary+enamel+knot+determines+the+position+of+the+first+buccal+cusp+in+developing+mice+molars.">The primary enamel knot determines the position of the first buccal cusp in developing mice molars.</a> Differentiation; research in biological diversity. 75, 441-451 (2007).</li><li>Sakano, M., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cell+dynamics+in+cervical+loop+epithelium+during+transition+from+crown+to+root:+implications+for+Hertwig's+epithelial+root+sheath+formation.">Cell dynamics in cervical loop epithelium during transition from crown to root: implications for Hertwig's epithelial root sheath formation.</a> Journal of periodontal research. , (2012).</li><li>Fisher, A. 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V. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Regeneration+of+halved+embryonic+lower+first+mouse+molars:+correlation+with+the+distribution+pattern+of+non+dividing+IDE+cells,+the+putative+organizers+of+morphogenetic+units,+the+cusps.">Regeneration of halved embryonic lower first mouse molars: correlation with the distribution pattern of non dividing IDE cells, the putative organizers of morphogenetic units, the cusps.</a> The International journal of developmental biology. 44, 289-295 (2000).</li><li>Kavanagh, K. D., Evans, A. R., Jernvall, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Predicting+evolutionary+patterns+of+mammalian+teeth+from+development.">Predicting evolutionary patterns of mammalian teeth from development.</a> Nature. 449, 427-432 (2007).</li><li>Munne, P. 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The authors declare that they have no competing financial interests.

This method of tooth culture has the advantage that access to the tooth germ is excellent, allowing accurate lineage tracing and placement of beads within the epithelium or the mesenchyme. Defined regions of the developing tooth germ can therefore be specifically targeted. During culture the changing morphology of the tooth germ can be followed, and the effect of manipulations quickly assessed.
The method, however, is only suitable for young tooth germs before substantial formation of hard tis...

For a number of experiments it is important to be able to culture tissue ex vivo to follow development. Culture of developing tissue provides access at defined periods of development and allows manipulation of genes by the addition of factors to the culture medium, or on loaded beads, and by the use of transfection and electroporation1. For many experiments it is important to be able to visualize the tissue as it grows, for example, to follow the fate of lineage labeled cells as the tissue undergoes morphogenesis. This can be particularly problematic for tissues that develop deep within the embryo, which are not obvious when a block of tissue from the embryo is cultured. Teeth are good examples of this, as they develop within the mandible, maxilla and frontal nasal process. When the whole mandible is cultured the superficial structures of the tooth can be viewed but changes in morphology can only be analyzed after sectioning of fixed tissue2. We have adapted a live slice culture technique allowing us to follow tooth germ development and providing access to the different parts of the tooth during development. The technique cultures the tooth germ slices at the gas-liquid interface, using a modified Trowell method3. These slice cultures have been very useful in directly following morphogenesis of the tooth, and allowing lineage tracing of distinct components, such as the enamel knot and the dental papilla and follicle1,4-7. The technique is not limited to mouse embryos and has successfully been used to culture live slices of pig and snake dental tissue8,9. In addition to the benefit of being able to visualize tooth development, the slice method also has the advantage that the thin slices of tissue have increased access to nutrients from the medium and air from the incubator. This results in improved growth of the tooth germs, which match development in vivo, and show invasion of endothelial cells into the papilla7. In contrast, tooth germs in whole mandible cultures develop slower than those in vivo and the center of the culture is often necrotic in long-term cultures. In slice culture, the tooth develops within a slice of the jaw, and its interaction with the surrounding developing bone and other tissues can be monitored. In our method the tissue is chopped straight after dissection with no need to embed in a support medium10,11&#160;and no need for any system to attach the tissue to the chopping block. The method is therefore noninvasive and rapid, allowing many mandibles to be sectioned in one session.

<table><tbody><tr><td>Ethanol</td><td>VWR</td><td>101077Y</td><td>100% ethanol was diluted in distilled H&lt;sub&gt;2&lt;/sub&gt;O to 70%.</td></tr><tr><td>DMEM F12</td><td>Gibco</td><td>12634-010</td><td>Advanced Dulbecco&#039;s Modified Eagle Medium F12</td></tr><tr><td>GlutaMAX</td><td>Gibco</td><td>35050-061</td><td></td></tr><tr><td>Penicillin-streptomycin</td><td>Sigma</td><td>P0781</td><td></td></tr><tr><td>DiI (Molecular probes)</td><td>Vybrant</td><td>V-22885</td><td>Cell-labeling solution</td></tr><tr><td></td><td>Invitrogen</td><td></td><td>Cell tracker CM-DiI, C-7000</td></tr><tr><td>DiO (Molecular probes)</td><td>Vybrant</td><td>V22886</td><td></td></tr><tr><td>Geminator 500</td><td>Thomas</td><td>Thomas No. 3885A20</td><td>Dry-heat sterilization</td></tr><tr><td>McIlwain tissue chopper</td><td>Ted Pella, Inc.</td><td>10180</td><td>Standard table</td></tr><tr><td>Organ culture dish (Center-Well Organ Culture Dish)</td><td>Falcon</td><td>353037</td><td></td></tr><tr><td>Membranes (Cell Culture Inserts, 0.4 &amp;mu;m pore size)</td><td>BD Falcon</td><td>353090</td><td>PET track-etched membrane, 6-well format</td></tr><tr><td>Metal grids (Stainless Steel - AISI 304 - Mesh)</td><td>Goodfellow</td><td>FE228710</td><td>(Fe/Cr18/Ni10)</td></tr><tr><td>AutoFlow Direct Heat CO&lt;sub&gt;2&lt;/sub&gt; Incubator</td><td>Nuaire</td><td>NU-5500</td><td></td></tr><tr><td>Picospritzer III</td><td>Intracel Ltd</td><td>051-0500-900 0-100 psi</td><td>Single channel picospritzer III</td></tr><tr><td>Glass capillary with filament 1 mm</td><td>WPI</td><td>TW100F-4</td><td></td></tr><tr><td>Tungsten wire 0.1 mm</td><td>Goodfellow</td><td>W005138</td><td></td></tr><tr><td>Tungsten wire 0.38 mm</td><td>Goodfellow</td><td>W005155</td><td></td></tr><tr><td>Aspirator tubes</td><td>Sigma</td><td>A5177</td><td>Used for mouth aspiration lineage tracers</td></tr></tbody></table>

the slice culture method for following development of tooth germs in explant culture

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental biologist. For many organs it is difficult to access developing tissue to allow monitoring during ex vivo culture. The slice culture method allows access to tissue so that morphogenetic movements can be followed and specific cell populations can be targeted for manipulation or lineage tracing.
In this paper we describe a method of slice culture that has been very successful for culture of tooth germs in a range of species. The method provides excellent access to the tooth germs, which develop at a similar rate to that observed in vivo, surrounded by the other jaw tissues. This allows tissue interactions between the tooth and surrounding tissue to be monitored. Although this paper concentrates on tooth germs, the same protocol can be applied to follow development of a number of other organs, such as salivary glands, Meckel&#39;s cartilage, nasal glands, tongue, and ear.

In order to follow the movement of the first molar dental follicle, 250 &#956;m frontal slices were taken through a mandible using the method described above. The dental follicle is the layer of mesenchyme that surrounds the outer enamel epithelium (OEE) of the developing tooth, and has previously been shown to take part in the formation of tissue of the periodontium 6. Mandibles were dissected at E14.5, the cap stage of tooth development. In the slice the outline of the dental epithelium was clear, and the co...

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

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.

Explant culture allows manipulation of developing organs at specific time points&#160;and is therefore an important method for the developmental ...

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14.0K Views.  King's College London. 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.

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

Migratory Behavior of Cells Generated in Ganglionic Eminence Cultures

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral cortex consists of two basic neuronal types: excitatory and inhibitory. These cells arise in distinct areas and migrate into the cortex along different routes (Pearlman et al., '98). Inhibitory interneurons migrate tangentially from subcortical sources, mostly from different regions of the ganglionic eminences (Gelman et al., '09; Xu et al., '04). Their movement requires precise spatiotemporal control imposed by environmental cues, to allow for the establishment of proper cytoarchitecture and connectivity in the cerebral cortex (Caviness &amp; Rakic, '78; Hatten, '90; Rakic, '90). To study the migratory behavior of cells generated in proliferative zones of the ganglionic eminences (GE) in newborn ferrets in vitro we used a 3 dimensional culture arrangement in a BD Matrigel Matrix. The culture setup consisted of two GE explants and a source of tested proteins extracted from the cerebral cortex and adsorbed on fluorescent latex Retrobeads IX positioned between the explants (Hasling et al., '03; Riddle et al., '97). After 2-3 days of culture, the cells start to appear at the edge of the explant showing a propensity to leave the tissue in a radial direction. Live imaging allowed observation of migratory patterns without the necessity of labeling or marking the cells. When exposed to fractions of the protein extract obtained from isochronic ferret cortex, the GE cells displayed different behaviors as judged by quantitative kinetic analysis of individual moving cells.

Time lapse imaging of 3D tissue culture allows studying migratory behavior of individual cells originating from ganglionic eminence in reaction to fractionated protein extract from cerebral cortex.

Migration of cells is a common process that leads to the development and maturation of the vertebrate central nervous system (Hatten, '99). The cerebral ...

Neuroscience

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

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. Without sensory innervation, daily oral activities would cause irreparable damage. Despite its importance, the roles of innervation in tooth development and maintenance have been largely overlooked. Several studies have demonstrated that DP cells secrete extracellular matrix proteins and paracrine signals to attract and guide TG axons into and throughout the tooth. However, few studies have provided detailed insight into the crosstalk between the DP mesenchyme and neuronal afferents. To address this gap in knowledge, researchers have begun to utilize co-cultures and a variety of techniques to investigate these interactions. Here, we demonstrate the multiple steps involved in co-culturing primary DP cells with TG neurons dispersed on an overlying transwell filter with large diameter pores to allow axonal growth through the pores. Primary DP cells with the gene of interest flanked by loxP sites were utilized to facilitate gene deletion using an Adenovirus-Cre-GFP recombinase system. Using TG neurons from the Thy1-YFP mouse allowed for precise afferent imaging, with expression well above background levels by confocal microscopy. The DP responses can be investigated via protein or RNA collection and analysis, or alternatively, through immunofluorescent staining of DP cells plated on removable glass coverslips. Media can be analyzed using techniques such as proteomic analyses, although this will require albumin depletion due to the presence of fetal bovine serum in the media. This protocol provides a simple method that can be manipulated to study the morphological, genetic, and cytoskeletal responses of TG neurons and DP cells in response to the controlled environment of a co-culture assay.

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.

Tooth innervation allows teeth to sense pressure, temperature and inflammation, all of which are crucial to the use and maintenance of the tooth organ. ...

Developmental Biology

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

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The continuously growing mouse incisor provides an excellent model to study tissue-specific stem cells. The epithelial tissue-specific stem cells of the incisor are located at the proximal end of the tooth in a niche called the cervical loop. They provide a continuous influx of cells to counterbalance the constant abrasion of the self-sharpening tip of the tooth. Presented here is a detailed protocol for the isolation and culture of the proximal end of the mouse incisor that houses stem cells and their niche. This is a modified Trowell-type organ culture protocol that enables in vitro culture of tissue pieces (explants), as well as the thick tissue slices at the liquid/air interface on a filter supported by a metal grid. The organ culture protocol described here enables tissue manipulations not feasible in vivo, and when combined with the use of a fluorescent reporter(s), it provides a platform for the identification and tracking of discrete cell populations in live tissues over time, including stem cells. Various regulatory molecules and pharmacological compounds can be tested in this system for their effect on stem cells and their niches. This ultimately provides a valuable tool to study stem cell regulation and maintenance.

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.

Organ development, function, and regeneration depend on stem cells, which reside within discrete anatomical spaces called stem cell niches. The ...

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.

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 ...

Live Tracking of Dental Cells via Two-Photon Microscopy

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

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal stem cells and tooth regeneration. These progenitor populations actively divide, move, and differentiate to maintain tissue homeostasis and regenerate lost cells in a responsive manner. However, traditional analyses using fixed tissue sections could not capture the dynamic processes of cellular movements and interactions, limiting our ability to study their regulations. This paper describes a protocol to maintain whole mouse incisors in an explant culture system and live-track dental epithelial cells using multiphoton timelapse microscopy. This technique adds to our existing toolbox for dental research and allows investigators to acquire spatiotemporal information on cell behaviors and organizations in a living tissue. We anticipate that this methodology will help researchers further explore mechanisms that control the dynamic cellular processes taking place during both dental renewal and regeneration.

Author Spotlight: Understanding Dynamic Cellular Behaviors in Adult Mouse Dental Tissue Renewal and Repairment 

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.

The continuously growing mouse incisor is emerging as a highly tractable model system to investigate the regulation of adult epithelial and mesenchymal ...

An Explant Assay for Assessing Cellular Behavior of the Cranial Mesenchyme

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the neural tube in a process known as neurulation. During neurulation, morphogenesis of the mesenchyme that underlies the neural plate is believed to drive neural fold elevation. The cranial mesenchyme is comprised of the paraxial mesoderm and neural crest cells. The cells of the cranial mesenchyme form a pourous meshwork composed of stellate shaped cells and intermingling extracellular matrix (ECM) strands that support the neural folds. During neurulation, the cranial mesenchyme undergoes stereotypical rearrangements resulting in its expansion and these movements are believed to provide a driving force for neural fold elevation. However, the pathways and cellular behaviors that drive cranial mesenchyme morphogenesis remain poorly studied. Interactions between the ECM and the cells of the cranial mesenchyme underly these cell behaviors. Here we describe a simple ex vivo explant assay devised to characterize the behaviors of these cells. This assay is amendable to pharmacological manipulations to dissect the signaling pathways involved and live imaging analyses to further characterize the behavior of these cells. We present a representative experiment demonstrating the utility of this assay in characterizing the migratory properties of the cranial mesenchyme on a variety of ECM components.

The cranial mesenchyme undergoes dramatic morphogenic movements that likely provides a driving force for elevation of the neural folds1,2. Here we describe a simple ex vivo explant assay to characterize the cellular behaviors of the cranial mesenchyme during neurulation. This assay has numerous applications including being amenable to pharmacological manipulations and live imaging analyses.

The central nervous system is derived from the neural plate that undergoes a series of complex morphogenetic movements resulting in formation of the ...

A Novel Method for the Culture and Polarized Stimulation of Human Intestinal Mucosa Explants

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper homeostasis directly depends on these interactions, as they shape an entire immunological response inducing tolerance against food antigens while at the same time mounting effective immune responses against pathogenic microbes accidentally ingested with food.
Intestinal homeostasis is preserved also through various complex interactions between the microbiota (including food-associated beneficial bacterial strains) and the host, that regulate the attachment/degradation of mucus, the production of antimicrobial peptides by the epithelial barrier, and the "education" of epithelial cells' that controls the tolerogenic or immunogenic phenotype of unique, gut-resident lymphoid cells' populations. These interactions have been so far very difficult to reproduce with in vitro assays using either cultured cell lines or peripheral blood mononuclear cells. In addition, mouse models differ substantially in components of the intestinal mucosa (mucus layer organization, commensal bacteria community) with respect to the human gut. Thus, studies of a variety of treatments to be brought in the clinics for important stress-related or pathological conditions such as irritable bowel syndrome, inflammatory bowel disease or colorectal cancer have been difficult to carry out.
To address these issues, we developed a novel system that enables us to stimulate explants of human intestinal mucosa that retain their in situ conditioning by the host microbiota and immune response, in a polarized fashion. Polarized apical stimulation is of great importance for the outcome of the elicited immune response. It has been repeatedly shown that the same stimuli can produce completely different responses when they bypass the apical face of the intestinal epithelium, stimulating epithelial cells basolaterally or coming into direct contact with lamina propria components, switching the phenotype from tolerogenic to immunogenic and causing unnecessary and excessive inflammation in the area.
We achieved polarized stimulation by gluing a cave cylinder which delimited the area of stimulation on the apical face of the mucosa as will be described in the protocol. We used this model to examine, among others, differential effects of three different Lactobacilli strains. We show that this model system is very powerful to assess the immunomodulatory properties of probiotics in healthy and disease conditions.

We introduce a novel method for the maintenance of human intestinal mucosa in culture and monitoring of the response to various types of stimuli over at least 24 hrs. With our method, the polarity of the tissue is maintained, allowing for a physiological stimulation via the apical route.

Few models currently exist to realistically simulate the complex human intestine's micro-environment, where a variety of interactions take place. Proper ...

Establishing Stable Binary Cultures of Symbiotic Saccharibacteria from the Oral Cavity

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial diversity on earth. Novel approaches are required to culture these uncultured bacteria so that investigators can effectively study their physiology and lifestyle using the powerful tools available in the laboratory. The Candidate Phyla Radiation (CPR) is one of the largest groups of uncultivated bacteria, comprising ~15% of the living diversity on earth. The first isolate of this group was a member of the Saccharibacteria phylum, 'Nanosynbacter lyticus' strain TM7x. TM7x is an unusually small bacterium that lives as a symbiont in direct contact with a bacterial host, Schaalia odontolytica, strain XH001. Taking advantage of the unusually small cell size and its lifestyle as a symbiotic organism, we developed a protocol to rapidly culture Saccharibacteria from dental plaque. This protocol will show how to filter a suspension of dental plaque through a 0.2 &#181;m filter, then concentrate the collected Saccharibacteria cells and infect a culture of host organisms. The resulting coculture can be passaged as any normal bacterial culture and infection is confirmed by PCR. The resulting binary culture can be maintained in the laboratory and used for future experiments. While contamination is a possibility, the binary culture can be purified by either further filtering and reinfection of host, or by plating the binary culture and screening for infected colonies. We hope this protocol can be expanded to other sample types and environments, leading to the cultivation of many more species in the CPR.

We demonstrate a method for isolating difficult-to-grow members of the novel bacterial phylum, Saccharibacteria, by filtering dental plaque and co-culturing with host bacteria.

Many bacterial species cannot be cultured in the laboratory using standard methods, posing a significant barrier to studying the majority of microbial ...

Explant Culture for Developmental Studies

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo approach allows researchers to manipulate and observe developing tissues in ways that are not possible in vivo. Once established, explant culture is frequently used to understand the role of genes and signaling molecules in organogenesis.
This video will first introduce the basic principles of explant culture and demonstrate a protocol to isolate and grow explanted mammalian tissues. Common genetic and molecular methods of manipulating explant cultures will then be discussed. Finally, the viewers will learn about how explant techniques are currently being applied to study organ development.

Explant Culture for Developmental Studies and Morphogenesis

Explant culture is a technique in which living cells or tissues are removed from an embryo for continued development outside of the organism. This ex vivo ...

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Explant Culture of Neural Tissue

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and changes in cell morphology. Studying these processes is essential to our understanding of nervous system function as well as our ability to diagnose and treat disorders that result from abnormal development. However, neural tissues are relatively inaccessible for experimental manipulations, especially in embryonic mammals. As a result, many scientists take advantage of explant culture in order to study neurodevelopmental processes in an &#8220;organotypic&#8221; environment, meaning that the tissue is removed from the organism but its complex cellular architecture is maintained. Generally, explant cultures are created by careful dissection of neural tissue that is then submerged in carefully designed growth media and cultured in vitro.
This video will first provide a brief overview of neural explant culture, including its advantages over other in vitro methods and important considerations for maintaining healthy tissue. Next, a general protocol will be provided for setting up an explant culture from embryonic mouse brain, outlining the isolation of embryos from the mother and dissection of the brain. The presentation also includes an overview of slice culture, in which thin sections of nervous system tissue are generated for improved visual access to the developing cells. Lastly, a few applications of these techniques will be provided to demonstrate how they can be used to answer important questions in the neurodevelopmental field.

The intricate structure of the vertebrate nervous system arises from a complex series of events involving cell differentiation, cell migration, and ...