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 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.
Over the past two decades, the mouse incisor has emerged as an invaluable platform for investigating the principles of adult stem cell regulation and tooth regeneration1,2. The mouse incisor grows continuously and renews itself throughout the animal's life. It does so by maintaining both epithelial and mesenchymal stem cells, which can self-renew and differentiate into different cell types of the tooth1,2. While dental epithelial stem cells give rise to ameloblasts, which secrete the enamel matrix, dental mesenchymal stem cells give rise to odontoblasts, cementoblasts, and fibroblasts, which form dentin, cementum, and periodontal ligament, respectively3,4,5,6. This constant supply of new cells maintains tissue homeostasis and allows the replacement of old cells that are lost due to masticatory wear or injuries7,8. Elucidating the cellular and molecular mechanisms that regulate the maintenance and differentiation of dental stem cells is therefore central to understanding dental regeneration, an area of growing interest.
Anatomically, a large portion of the adult mouse incisor is encased in the jawbone. While the incisal edge of the tooth is exposed, the apical end of the incisor fits within a socket and is firmly attached to the surrounding bone through periodontal ligaments and connective tissues (Figure 1A,B). The incisor's apical end is also the growth region of the tooth and maintains dental stem and progenitor cells in both the epithelial layer and the mesenchymal pulp9,10,11,12,13. Specifically, dental epithelial stem cells are maintained at the bulbous end of the epithelium, known as the apical bud, also referred to as the labial cervical loop (Figure 1C). Similar to the intestinal epithelium and the epidermis, epithelial renewal in the incisor is primarily supported by actively cycling stem cells and their highly proliferative intermediate descendants, called transit-amplifying cells14,15,16,17, both residing in the inner part of the cervical loop. However, whether the incisor epithelium contains and utilizes quiescent stem cells during regeneration remains to be determined. In contrast, both active and quiescent dental mesenchymal stem cells have been identified in the apical pulp, and the quiescent stem cells function as a reserve population that becomes activated during injury repair13,18.
Many of the discoveries on the biology of the mouse incisor renewal and regeneration have resulted from histological investigations, in which samples are obtained at distinct temporal junctures, fixed, processed, and then sectioned into micron-thin slices along a particular plane. Through detailed analysis of histological sections from different mouse models that enable lineage tracing or genetic perturbations, scientists have identified the cell lineages of different progenitor populations, as well as the genetic and signaling pathways that control incisor homeostasis and injury repair19,20,21. However, the static two-dimensional (2D) images of non-vital cells in sections cannot capture the full spectrum of cellular behaviors and spatial organizations in living tissue, such as cell shape changes, movements, and cellular kinetics. Detecting and measuring these rapid cellular changes, which occur at a timescale that is unresolvable through tissue sectioning, require a different strategy. Moreover, acquiring such information is also critical for understanding how dental cells interact with each other, react to different signaling stimuli, and self-organize to maintain tissue structures and functions.
The advent of four-dimensional (4D) deep tissue imaging using two-photon microscopy22, a technology that integrates three spatial dimensions with temporal resolution, overcomes the inherent limitations of histological analysis by enabling spatiotemporal examination of cultured tissue explants, organoids, or even tissues in situ23,24,25,26. For instance, 4D live imaging of the developing tooth epithelium has unveiled the spatiotemporal patterns of cell divisions and migrations that coordinate tissue growth, signaling center formation, and dental epithelial morphogenesis27,28,29,30,31,32. In the adult mouse incisor, 4D imaging has been recently adapted to study cellular behaviors during dental epithelial injury repair. Live imaging revealed that stratum intermedium cells in the suprabasal layer can be directly converted into ameloblasts in the basal layer to regenerate the damaged epithelium, challenging the traditional paradigm of epithelial injury repair15.
Here, we describe the dissection, culturing, and imaging of the adult mouse incisor, focusing on epithelial cells in the labial cervical loop (Figure 1). This technique preserves dental cell vitality for more than 12 h and permits live tracking of fluorescently labeled cells at single-cell resolution. This approach allows investigation of cell motion and migration as well as dynamic changes in cell shape and division orientation under normal culture conditions, or in responses to genetic, physical, and chemical perturbations.
All mice were maintained in pathogen-free animal facilities at the University of California Los Angeles (UCLA) or the Hebrew University of Jerusalem (HUJI). All experiments involving mice were performed according to regulations and protocols approved by the respective Institutional Animal Care and Use Committee (IACUC) (ARC-2019-013; UCLA) or (MD-23-17184-3; HUJI). A general workflow of the experimental steps is shown in Figure 2A. See the Table of Materials for details related to all instruments, reagents, and materials used in this protocol.
1. Preparation of solutions and gels
2. Extraction of the adult mouse mandibles
3. Isolation of the whole mouse incisors
NOTE: Further isolation of the incisor is done under a bright field dissection microscope.
4. Removal of periodontal tissues to expose the incisor epithelial cervical loop
5. Tissue embedding for explant culture
6. Timelapse microscopy of incisor explants
NOTE: In this experiment, we used an upright microscope equipped with a 25x water-dipping objective that has a numerical aperture of 1. In general, a water dipping lens with a high numerical aperture is best suited for deep tissue imaging.
The apical region of the adult mouse incisor is encased within the mandible (Figure 1) and hence, not directly accessible for visualizing and live-tracking the progenitor cells residing within the growth region. Therefore, we have developed a method to extract the whole incisor from the jawbone and maintain it in an explant culture system for two-photon timelapse microscopy (Figure 2). Here we describe representative results that capture the dynamic process of cell proliferation and movement in the labial cervical loop region of the dental epithelium.
To demonstrate the experimental procedures, we have used two different mouse models that express green fluorescence in the dental epithelium. The first mouse line is K14Cre;R26rtTA;tetO-H2B-GFP, where K14Cre (MGI:2680713) expresses the Cre recombinase from a Keratin 14 promoter35 and activates the expression of the reverse tetracycline-controlled transactivator in the epithelium from the R26rtTA allele36 (MGI:3584524). Upon administration of doxycycline, rtTA induces histone H2B-GFP expression from the tetO-H2B-GFP allele37 (MGI:3043783) and labels epithelial cell nuclei with green fluorescence. This is especially useful for cell tracking and for detecting cell divisions. In this experiment, we fed animals with doxycycline food for 24 h before sacrifice to activate H2B-GFP expression. The second mouse line is K14Cre;R26mT/mG, in which R26mT/mG (MGI:3803814) is a Cre-reporter38. In the absence of Cre activity, cells express membrane-localized tdTomato (mT) with red fluorescence. Upon Cre-mediated recombination, cells express membrane GFP (mG). K14Cre;R26mT/mG thus labels epithelial cell membranes with green fluorescence, leaving non-epithelial cells red. This permits easy visualization of cell shapes, divisions, and movements.
We began the procedure by dissecting out the mandibles (Figure 3A) and then systematically removed all the bones surrounding the incisor (Figure 3B–G). This yielded whole incisors with undamaged epithelium (Figure 3H). We confirmed the intactness of the dental epithelium by inspecting the green fluorescence in the K14Cre;R26rtTA;tetO-H2B-GFP and K14Cre;R26mT/mG mice (Figure 4A,B,E,F). At this stage, the opaque periodontal tissues still cover the apical incisor, and the cervical loop thus appears blurry due to light scattering, which would similarly hinder downstream timelapse imaging (Figure 4C,G). We therefore carefully removed the periodontal tissues, so the dental epithelium with the cervical loop could be discerned in each incisor (Figure 4D,H).
The incisors were then embedded in low melting point agarose and cultured in a perfusion setup for two-photon live imaging as depicted in Figure 2. For this exercise, we have focused on the cervical loop region of the dental epithelium and captured z-stack images at 4 µm intervals every 5 min over a duration of 14 h (Figure 5A). Notably, H2B-GFP signals were mostly observed in the transit-amplifying region of the cervical loop, where there are active cell divisions (Figure 5B). This is likely because there is higher H2B-GFP exchange at open chromatins and incorporation into nucleosomes following DNA replications in these active cells39.
We subsequently examined the timelapse images using ImageJ and based on the separating of the H2B-GFP signals40, we were able to observe numerous cell divisions throughout the imaging period (Supplemental Video S1 and Supplemental Video S2). This indicated that the tissues were adequately maintained in the explant culture and cells were active. Specifically, we were able to observe the condensation and alignment of chromosomes at the metaphase plate in mitotic cells, followed by their segregation into two daughter cells during anaphase (Figure 5C–N, manually tracked using the ImageJ plugin TrackMate). Most of these divisions were perpendicular or at an oblique angle relative to the basement membrane (Figure 5C–K and Supplemental Video S1). Horizontal divisions parallel to the basement membrane could also be detected, although occurring less frequently (Figure 5L–N and Supplemental Video S2). It is important to point out that cytokinesis in the dental epithelium often happens quickly, within 5-10 min. As a result, timelapse intervals of more than 5 min may miss some of these divisions.
Cell division events were also apparent in K14Cre;R26mT/mG cervical loops, where all epithelial cell membranes were labeled green (Figure 6A). We could identify mitotic cells by their cell rounding and then cytokinesis (Supplemental Video S3), and both vertical and horizontal cell divisions were observable (Figure 6B–G), thus similar to the results obtained using H2B-GFP. Together, these results demonstrate that this protocol can serve as a powerful tool to investigate cell behaviors in the incisor explants when combined with mouse genetic models that fluorescently label distinct subcellular structures.
Figure 1: Schematics of the mouse jaw and incisor cervical loop. (A) A significant portion of the incisor is embedded in the jawbone. The growth region is located at the apical end of the tooth and supports its continuous growth (dark green arrow). (B) An enlargement of the apical incisor, which is surrounded by periodontal tissues. The tooth is composed of enamel and dentin, which are highly mineralized structures formed by ameloblasts and odontoblasts, respectively. (C) A sagittal section of the apical incisor, showing that dental epithelial progenitor cells and transit-amplifying cells reside in the labial cervical loop and give rise to ameloblasts in the more distal epithelium (dark green dashed arrow). Compared to the labial cervical loop, the lingual cervical loop is smaller in size and does not normally form ameloblasts. Dental mesenchymal stem cells are present in the dental pulp (purple region) and give rise to odontoblasts. Abbreviations: En = enamel; De = dentin; Am = ameloblast; Od = odontoblast; TACs = transit-amplifying cells; laCL = labial cervical loop; liCL = lingual cervical loop. Please click here to view a larger version of this figure.
Figure 2: Maintaining mouse incisor explant for live imaging. (A) Schematics depicting key steps of the protocol, from dissecting the incisor to embedding the tissue in low melting point agarose for live imaging. A perfusion setup is used to provide a constant supply of nutrients during imaging and the culture is maintained at 37 °C. (B–D) A step-by-step demonstration of setting the culture dish and the perfusion chamber for live imaging. The media inlet and outlet are shown as pink and yellow arrows respectively. Abbreviation: ACBR = Atmospheric Control Barrier Ring. Please click here to view a larger version of this figure.
Figure 3: Isolation of the whole mouse incisor. (A) Intact jaw. (B–H) Bones were gradually removed to expose the whole incisor. Red dashed lines in B and C show the exposed soft tissue of the apical incisor after shaving off the membrane bone overlying the lingual and buccal sides of the incisor socket (steps 3.3-3.5 in the protocol). (D–G) Blue arrowheads represent the condyles, molars, and alveolar bones that have been removed to isolate the entire tooth (step 3.6 in the protocol). Scale bar = 2 mm (H). Please click here to view a larger version of this figure.
Figure 4: Removal of periodontal tissues for live imaging. (A–H) Representative samples from (A–D) K14Cre;R26rtTA;tetO-H2B-GFP,and (E–H) K14Cre;R26mT/mGmice were used in our demonstration of the protocol. Before dissection, GFP expression in the dental epithelium was visible through the bone (A,E, yellow arrowheads). White dashed lines outline the undissected incisors. E' shows the lingual side. In isolated incisors (B,C,F,G), GFP fluorescence was initially diffracted by the periodontal tissues covering the apical incisors (white and red arrowheads). Red fluorescence in F and G labels non-epithelial cells. Removal of periodontal tissues allows a clear and unobstructed view of the green fluorescent cervical loops (D,H, green arrowheads). Scale bar = 2 mm (A,E); 1.25 mm (B,F); and 300 µm (C,D,G,H). Please click here to view a larger version of this figure.
Figure 5: Timelapse microscopy of the K14Cre;R26rtTA;tetO-H2B-GFP cervical loop. (A) A schematic illustrating the timelapse setup. (B) A representative z-plane of the K14Cre;R26rtTA;tetO-H2B-GFP incisor labial cervical loop, showing nuclear labeling by H2B-GFP primarily in the transit-amplifying region, where cells actively divide. The yellow box represents the general area of the enlarged images shown below. (C–K) Timelapse images showing vertical and oblique cell divisions relative to the BM. (L–N) Timelapse images show an example of horizontal cell division relative to the BM. (D,G,J,M) Cells in metaphase or anaphase are displayed in the middle panels. Schematics of each tracked division are shown on the right. Scale bar in N = 36 µm (B); 5 µm (C–N). Abbreviation: BM = basement membrane. Please click here to view a larger version of this figure.
Figure 6: Timelapse microscopy of the K14Cre;R26mT/mG cervical loop. (A) A representative z-plane of the K14Cre;R26mT/mG incisor labial cervical loop. All epithelial cells express membrane GFP. The yellow box represents the area of cell tracking shown in the enlargements. (B–G) Timelapse images capturing both (B–D) horizontal and (E–G) vertical cell divisions, relative to the BM. (C,F) Middle panels show mitotic cell rounding. Schematics of each tracked division are shown on the right. Scale bar = 35 µm (A); 5 µm (B–G). Abbreviation: BM = basement membrane. Please click here to view a larger version of this figure.
Supplemental Figure S1: Inhouse-made perfusion dish. (A) A 35 mm culture dish can be used to make a perfusion dish. A glass bottom dish can be used if imaging is performed using an inverted microscope. (B) Heat a nail with a flame. (C) Two openings (white arrowheads) are created on either side of the dish using the heated nail. (D) Affix two blunt-end 16 G needles, one as the media inlet and the other as the media outlet, through the openings using epoxy glue. If gas perfusion is desired, a third opening/needle can be added to the dish. Please click here to download this File.
Supplemental Video S1: Live imaging of a K14Cre;R26rtTA;tetO-H2B-GFP incisor labial cervical loop, showing vertical and oblique cell divisions. Cells are manually tracked, and different colored dots represent different pairs of mother/daughter cells. Scale bar = 30 µm (left frame); 7 µm (right frame). Please click here to download this Video.
Supplemental Video S2: Live imaging of a K14Cre;R26rtTA;tetO-H2B-GFP incisor labial cervical loop, showing an example of horizontal cell division. The horizontal division takes place at the 10 s mark and is manually tracked using blue dots. Scale bar = 30 µm (left frame); 7 µm (right frame). Please click here to download this Video.
Supplemental Video S3: Live imaging of a K14Cre;R26mT/mG incisor labial cervical loop, showing both vertical and horizontal cell divisions. Cells are manually tracked, and different colored circles represent different pairs of mother/daughter cells. Scale bar = 30 µm (left frame); 7 µm (right frame). Please click here to download this Video.
Live tissue imaging is an important technique that allows us to study the dynamic processes and behaviors of cells when they are maintained in their niche environment41. Ideally, live imaging is performed in vivo with high spatiotemporal resolution. However, in vivo imaging for mammalian organs can be challenging due to tissue inaccessibility, optical opaqueness, and difficulty in immobilizing the animal or the organ for a prolonged period42. Tissue explants bypass some of these challenges and have been successfully adopted in a number of studies to track cell behaviors, such as during tooth morphogenesis and the development of ectodermal organs26. Here, we demonstrated a relatively simple yet robust protocol to perform live deep tissue imaging on cultured whole adult mouse incisors. Application of this technique will help us understand the regulation of cell movements, fate decisions, and tissue organizations during dental regeneration.
Because cell divisions are easily observable events and characteristic of regenerating tissues, we have focused on tracking dividing cells in this study to highlight one usage of the protocol. We found that incisor epithelial progenitors can undergo both vertical and horizontal divisions, thus similar to other epithelial tissues43. Elucidating the mechanism that regulates division angles and investigating the role of division orientations in cell fate decisions will be of importance in future studies. Combining live imaging with different mouse genetic models that also carry fluorescent cell fate markers, signaling pathway reporters, or the Fucci cell cycle reporter44,45 will help unveil how cell division patterns and cell cycle dynamics contribute to the regulation of epithelial homeostasis and regeneration.
A necessary practice in this protocol is to carry out tooth dissection in an expeditious and careful manner, allowing quick transition to and preservation of the intact tissue under the appropriate culturing condition. We have found that for live imaging, dissecting tissues in warm media, rather than cold media, as in experiments aimed to extract proteins and mRNAs46, maintains cells in a functioning state. Because the incisor is a relatively large organ for culturing, a steady supply of nutrients via media perfusion is also critical for the sustainment of the tissue throughout imaging47. If the progenitor cells are present in the region of interest, observation of frequent cell divisions is a good indicator that the tissue is healthy and active. In contrast, cell death should be infrequent, and this can be verified by detecting bright and condensed nuclear bodies during imaging if a nuclear marker is used or by performing TUNEL staining after imaging and tissue fixation48. In addition to nutrients, oxygen levels may also affect explant viability, as either oxygen tension or insufficiency can disrupt cell survival, proliferation, and differentiation49,50,51. In our experience, we have not observed apparent differences in cell proliferation and cell death when samples were provided with or without additional oxygen (e.g., 95% O2/5% CO2 carbogen or 5% CO2/21% O2/balanced N2) during imaging, suggesting that either trace amount of oxygen in the system is sufficient to maintain incisor epithelia in culture overnight or the tissue can employ alternative pathways to maintain normal cell functions52. However, the requirement for oxygen in other cellular processes or correct gene expression should be further determined in future studies.
In our demonstration of the protocol, we have used genetically encoded H2B-GFP and membrane GFP to label cell nuclei and contours, respectively. These fluorescent markers are bright and long-lasting under two-photon microscopy and are thus examples of ideal labels to use for tracking cell movements and divisions. In theory, fluorescent vital dyes can also be used to label different subcellular compartments for ex vivo live imaging53, but reduced penetration of the dyes into deeper epithelial cells likely limits their uses. Similarly, while standard confocal microscopy is capable of high-resolution live imaging at a depth under 100 µm, two-photon microscopy provides increased imaging depth with reduced photobleaching and phototoxicity54. We have therefore chosen two-photon microscopy as an imaging modality in most of our live imaging applications.
One potential limitation of this protocol is that incisor explants may not fully recapitulate the in vivo environment and biology. Data therefore represent cell behaviors under the culture condition and should be interpreted accordingly as well as validated using other methods, such as histology, when applicable. We also had to partially remove periodontal tissues to gain access to the cervical loop for imaging. Signaling interactions between periodontal tissues and the dental epithelium could thus be disrupted. Lastly, while media perfusion greatly improves the viability of cultured tissues, it could introduce fluid shear stress that acts as an ectopic mechanical signal and affects results55,56. We have not determined the impact of different flow rates in our setup. However, because a low flow rate of 1-5 mL/h is typically sufficient for maintaining nutrient supplies57, the effect of shear stress can be minimized. With these limitations in mind, ex vivo live imaging serves as a powerful platform to study changes in cell behaviors when the appropriate controls are included in experiments.
The continuously growing mouse incisor is a highly tractable model system to study stem cell-based tissue renewal and injury repair. The protocol described herein provides investigators with the means to maintain whole adult incisors in culture and extract spatiotemporal information about cell behaviors through timelapse microscopy. We expect this technique to be broadly applicable to studying different mouse genetic models used in dental research and to help advance our knowledge in the field of dental regeneration.
The authors have nothing to disclose.
We acknowledge the UCLA Advanced Light Microscopy/Spectroscopy Laboratory and Leica Microsystems Center of Excellence at the California NanoSystems Institute (RRID:SCR_022789) for providing two-photon microscopy. AS was supported by ISF 604-21 from the Israel Science Foundation. JH was supported by R03DE030205 and R01DE030471 from the NIH/NIDCR. AS and JH were also supported by grant 2021007 from the United States-Israel Binational Science Foundation (BSF).
24 well, flat bottom tissue culture plate | Olympus plastics | 25-107 | |
25x HC IRAPO motCORR water dipping objective | Leica | 11507704 | |
Ascorbic acid (Vitamin C) | Acros Organics | 352685000 | |
D-(+)-Glucose bioxtra | Sigma Aldrich | G7528 | |
Delta T system | Bioptechs | 0420-4 | Including temperature control, culture dishes, and perfusion setup |
Dissection microscope- LEICA S9E | Leica | LED300 SLI | |
DMEM/F12 | Thermo Scientific | 11039047 | Basal media without phenol red |
Feather surgical blade (#15) | Feather | 72044-15 | |
Fine forceps | F.S.T | 11252-23 | |
Glutamax | Thermo Scientific | 35050-061 | Glutamine substitute |
Leica SP8-DIVE equipped with a 25X HC IRAPO motCORR water dipping objective | Leica | n/a | |
low-melting agarose | NuSieve | 50080 | |
non-essential amino acids (100x) | Thermo Scientific | 11140-050 | |
penicillin–streptomycin | Thermo Scientific | 15140122 | 10,000 U/mL |
Petri dish | Gen Clone | 32-107G | 90 mm |
Rat serum | Valley Biomedical | AS3061SC | Processed for live imaging |
Razor blade #9 | VWR | 55411-050 | |
Scalpel handle | F.S.T | 10003-12 | |
Scissors | F.S.T | 37133 | |
serrated forceps | F.S.T | 11000-13 | |
spring scissors | F.S.T | 91500-09 |