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 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's cartilage, nasal glands, tongue, and ear.
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 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.
1. Set Up
2. Embryo Dissection
3. Embryo Slicing
4. Slice Culture
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.
In order to follow the movement of the first molar dental follicle, 250 μm 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 condensing dental mesenchyme could be identified as a darker ring around the tooth epithelium (Figure 4A). DiI was injected in the mesenchyme next to the outer enamel epithelium of the tooth slice on the lingual side (Figure 4A). After labeling the slices were cultured for 4 days and photographed at intervals (Figures 4B and 4C). In vivo the tooth germs would have reached the bell stage by E18.5, and a distinct bell stage shape was observed in the slices, indicating a similar progression over this time period. The spot of DiI was seen to extend into a band of cells extending around the outer dental epithelium as the tooth grew (Figures 4B and 4C).
Figure 1. Formation of live slices. (A) E14.5 embryo. Dashed lines indicate the planes cut with a dissecting needle, starting with the lower cut for decapitation. (B) Dissected lower jaw. The tongue is facing downwards. The plane of section for the tissue chopper is shown using dashed lines. (C) Tissue chopper showing blade arm (BA) and white mounting disc (MD) with prepared specimen (arrow). (D) Representative slice through the molar region. Arrows point to molar tooth germs. (E) High power view of a molar slice. The tooth germ epithelium is outlined with black spots. Images sharpened using Photoshop. T = Tongue, DB = Dentary bone, MC = Meckel's cartilage. Scale bar in A = 3 mm. Scale bar in B & D = 1 mm. Scale bar in E = 500 μm. Click here to view larger figure.
Figure 2. Incisor and gland culture. (A) E12.5 Mandible that has been chopped sagittally. Developing incisors (outlined white spots with asterix) and submandibular glands (outlined with black spots and arrowed) can be just made out. (B) Same mandible showing central 4 slices separated out. The developing submandibular gland can be seen as a bud surrounded by condensed mesenchyme (arrowheads) in the two more lateral sections (top of image). The condensing mesenchyme around the gland is outlined in red. The incisors are at the epithelial thickening stage (asterix) in the two central slices (bottom of image). (C) Sagittal slice through an incisor at E14.0 at Day 0. (D) Same slice 1 day later. (E) Same slice after 3 days in culture. (C-E) Arrow points to incisor and forming labial cervical loop. Arrowhead points to developing submandibular gland. In culture the tooth germ extends backwards as the cervical loops grow, while the salivary gland continues to undergo branching morphogenesis and lumen formation. Images sharpened using Photoshop. T = Tongue, MC = Meckel's cartilage. Scale bar in A & B = 1 mm. Scale bar in C, D, E = 500 μm. Click here to view larger figure.
Figure 3. Culture method. (A) Cultured slice. The culture sits on a transparent filter suspended over medium via a metal grid. A hole in the grid allows the slice to be visualized with light from below. (B) Schematic of culture method.
Figure 4. DiI labeling of the dental follicle. (A-C) Merged light and dark field images showing developing molar tooth germ and location of DiI lineage label. (A) Day 0. The tooth germ (arrowhead) is at the cap stage. DiI labels cells of the dental follicle on the lingual side of the tooth. (B) Day 1. (C) Day 4. The tooth germ has reached the bell stage and the DiI labeled cells are seen to spread out in an arc around the developing outer enamel epithelium. Images have been sharpened in Photoshop after merging layers using the screen mode. Epithelium of tooth outlined with black spots. DP = dental papilla lying within the inner enamel epithelium. DF = dental follicle, which runs around the outer enamel epithelium. MC = Meckel's cartilage. Scale bar in A, B, C = 500 μm. Click here to view larger figure.
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 tissues, such as dentine and enamel, as these cannot be chopped accurately. For mandibles after E15.5 it is necessary to remove the bone before chopping. This has the disadvantage of potentially damaging the tooth germ, which is closely associated with the bone from E16.5. We have successfully sliced dissected tooth germs up to postnatal day 4, after which the tooth becomes too hard for the chopper to cut due to the deposition of enamel. The slice method works well on tooth germs from E13.5, i.e. the bud stage1,6. Before this time-point, when the tooth is at the epithelial thickening stage, the tooth germs do develop but the success rate is reduced and the morphology can be affected over the long term.
One major disadvantage with the method is that the chopping occurs at random through the tooth. In some slices a whole tooth germ will be found within a slice, while in others the chopper may cut the tooth germ through the middle. This means that it may be difficult to obtain large numbers of identical slices. To combat this problem, it is possible to divide a slice down the middle and use the right and left sides as experimental and control. For this, however, the mandible must be carefully placed on the chopping disc to ensure the slice is cut symmetrically. For some experiments it is an advantage to have slices that dissect the tooth so that internal structures, such as the enamel knot, can be accessed for lineage labeling4. The tooth germ appears very robust and such half tooth germs are able to develop well in culture, as previously shown in halved molar tooth germs12,13.
As an alternative to slice culture, tooth germs can be dissected out of the mandible and cultured in isolation14. This removes the problem of poor nutrition and oxygenation associated with culturing large blocks of tissue and leads to good tooth development but as a consequence the tooth develops without interaction with the surrounding tissue. When large amounts of the surrounding mesenchyme are removed the number of tooth germs that form can be altered, highlighting the importance of the surrounding mesenchyme for normal tooth development15. Slice culture is therefore a good method for studying the interactions of tissues, for example how the bone and tooth interact in the formation of the alveolar bone, or how the salivary glands interact with the tongue and oral epithelium, something that is lost when these tissues are cultured in isolation.
This paper highlights the use of this method for culturing the tooth germs but the same method is also excellent for culturing developing submandibular and sublingual glands and following the development of structure such as Meckel's cartilage (Figure 2).
The authors have nothing to disclose.
Sarah A. Alfaqeeh is funded by Kind Saud University College of Dentistry, Ministry of Higher Education, Kingdom of Saudi Arabia.
Name of Reagent/Material | Company | Catalog Number | Comments |
Ethanol | VWR | 101077Y | 100% ethanol was diluted in distilled H2O to 70%. |
DMEM F12 | Gibco | 12634-010 | Advanced Dulbecco's Modified Eagle Medium F12 |
GlutaMAX | Gibco | 35050-061 | |
Invitrogen | |||
Penicillin-streptomycin | Sigma | P0781 | |
DiI (Molecular probes) | Vybrant | V-22885 | Cell-labeling solution |
Invitrogen | Cell tracker CM-DiI, C-7000 | ||
DiO (Molecular probes) | Vybrant | V22886 | |
Invitrogen | |||
Geminator 500 | Thomas | Thomas No. 3885A20 | Dry-heat sterilization |
McIlwain tissue chopper | Ted Pella, Inc. | 10180 | Standard table |
Organ culture dish (Center-Well Organ Culture Dish) | Falcon | 353037 | |
Membranes (Cell Culture Inserts, 0.4 μm pore size) | BD Falcon | 353090 | PET track-etched membrane, 6-well format |
Metal grids (Stainless Steel – AISI 304 – Mesh) | Goodfellow | FE228710 | (Fe/Cr18/Ni10) |
AutoFlow Direct Heat CO2 Incubator | Nuaire | NU-5500 | |
Picospritzer III | Intracel Ltd | 051-0500-900 0-100 psi | Single channel picospritzer III |
Glass capillary with filament 1 mm | WPI | TW100F-4 | |
Tungsten wire 0.1 mm | Goodfellow | W005138 | |
Tungsten wire 0.38 mm | Goodfellow | W005155 | |
Aspirator tubes | Sigma | A5177 | Used for mouth aspiration lineage tracers |