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Published in final edited form as: J Tissue Eng Regen Med. 2014 Sep 4;11(3):618–626. doi: 10.1002/term.1952

Three-dimensional cultures of mouse submandibular and parotid glands: a comparative study

Noel J Leigh 1, Joel W Nelson 1, Rachel E Mellas 1, Andrew D McCall 2, Olga J Baker 1,*
PMCID: PMC4363090  NIHMSID: NIHMS669671  PMID: 25186108

Abstract

Freshly isolated salivary cells can be plated on an extracellular matrix, such as growth factor-reduced Matrigel (GFR-MG), to induce the formation of three-dimensional (3D) structures. Cells grown on GFR-MG are able to form round structures with hollow lumina, capable of sustaining amylase expression. In contrast, cells grown on plastic do not exhibit these features. Our recent studies have used mouse parotid gland (PG) cells, grown on different extracellular matrices, as a model for acinar formation. However, PG cells were not able to respond to the secretory agonist carbachol beyond 5 days and did not sustain polarity over time, regardless of the substratum. An alternative option relies in the use of mouse submandibular glands (SMG), which are more anatomically accessible and yield a larger number of cells. We compared SMG and PG cell clusters (partially dissociated glands) for their ability to form hollow round structures, sustain amylase and maintain secretory function when grown on GFR-MG. The results were as follows: (a) SMG cell clusters formed more organized and larger structures than PG cell clusters; (b) both SMG and PG cell clusters maintained α-amylase expression over time; (c) SMG cell clusters maintained agonist-induced secretory responses over time; and (d) SMG cell clusters maintained secretory granules and cell–cell junctions. These results indicate that mouse SMG cell clusters are more amenable for the development of a bioengineered salivary gland than PG cell clusters, as they form more organized and functional structures.

Keywords: salivary gland, epithelium, submandibular, parotid, matrigel and secretion

1. Introduction

Over the past 90 years of salivary research, many investigators have shown the classic submandibular branching morphogenesis as an ex vivo model for organ culture (Hieda et al., 1996; Hoffman et al., 2002; Koyama et al., 2003; Patel et al., 2006; Sakai et al., 2003). More recently, this organ culture has been used for transplantation in mice; however, these mice did not survive beyond 7 days (Ogawa et al., 2013). Other studies have shown that intraglandular transplantation of c-Kit+ cells into irreversibly damaged glands resulted in long-term restoration of salivary gland morphology and function in mice (Lombaert et al., 2008). Unfortunately, the self-renewal capacity of the salivary stem cells in this study was limited (Lombaert et al., 2008). A growing number of studies are currently characterizing salivary cell culture models towards the main goal of bioengineering a salivary gland (Lombaert et al., 2011; McCall et al., 2013; Pradhan-Bhatt et al., 2013, 2014). However, these models have failed in producing highly organized structures.

There are three pairs of major salivary glands in mice and humans, submandibular (SMG), sublingual (SLG) and parotid (PG) glands. In mice, the acini of SMG are seromucous, with additional secretory (serous) cells in a segment of the ducts called the granular convoluted tubules, SLG tubulo-acini are mucous with serous caps or demilunes, while PG acini are serous (Dobrosielski-Vergona, 1993). Our previous studies have used mouse PG as in vitro model to study salivary gland physiology and morphology. Specifically, we used growth factor-reduced Matrigel (GFR-MG) to study how salivary cells organize in a three-dimensional (3D) environment (McCall et al., 2013). The objective of this study was to compare the behaviour of mouse SMG and PG cell clusters when grown on GFR-MG, with the main goal of improving current conditions for salivary gland bioengineering.

2. Materials and methods

2.1. Experimental animals

Female C57BL/6 mice at age 5 weeks were anaesthetized using 80–100 mg/kg ketamine + 5 mg/kg xylazine. The mice were euthanized by either cervical dislocation, transcardial perfusion or abdominal exsanguination (Figures 1–3). SMGs and PGs were removed for the preparation of dispersed cell aggregates, as described below. All animal usage, anaesthesia and surgeries were conducted under the protocol and approval of the State University of New York at Buffalo Institutional Animal Care and Use Committee (IACUC).

Figure 1.

Figure 1

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Immunofluorescence of submandibular gland (SMG) and parotid gland (PG) cell clusters. Cell clusters from C57BL/6 mice SMG and PG were plated on growth factor-reduced Matrigel and allowed to grow for 0–14 days. Cells were fixed as described in Materials and methods. Localization of actin was determined using fluorescence microscopy, as follows: (A–F) phalloidin (actin), red; and (A–F) propidium iodide nuclear stain, blue; white arrows indicate F-actin ring and lumen formation

Figure 3.

Figure 3

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Agonist-stimulated intracellular calcium responses in SMG and PG cell clusters, which were plated on eight-well chambers and covered with growth factor-reduced Matrigel, then cell clusters were stimulated with Carbachol (100 μm). Changes in Fluo-2 fluorescence intensity were recorded as described in Materials and methods and analysed using Leica Application Suite Advanced Fluorescence software. Results from a representative experiment are shown (n≥3)

2.2. Salivary gland dissection

The skin between the end of the mandible and forelegs was tented and trimmed with surgical scissors. Under this incision, the two SMGs (which are attached to the sublingual glands) were exposed and removed. The skin was then trimmed towards the ear, revealing the PG and lacrimal gland. Directly ventral to the ear, on top of the masseter muscle, was the lacrimal gland (intersected with the craniofacial nerve pairs VII and IX). The PG was found posterior to the lacrimal gland, anterior to the forelegs, and was removed.

We found that salivary glands removed prior to abdominal exsanguination exhibit a large amount of blood in the glands, but this allows for better visualization of the different organs. We dissected a mouse using this methodology and were better able to distinguish the different glands based on their colour. The lacrimal gland has a yellow hue, SMG and SLG have a dark pink hue and the PG has a white hue. When the mouse was perfused with 2% w/v paraformaldehyde, the glands' natural colours were preserved; however, these cells are not suitable for cell culture. To avoid large amounts of blood or unsuitable cells, an alternative relies in the commonly used abdominal exsanguination (in which glands turn white). For this method, it is necessary to use a dissecting microscope (which helps in distinguishing the glands).

2.3. Whole mouse perfusion

Female C57BL/6 mice were anaesthetized and the thoracic cavity was opened by removal of the sternum. Sterile saline solution was perfused through the left ventricle at a pressure of 100 mmHg, following an incision to the right atrium. When the exiting fluid became clear, the perfusate was switched to 4% paraformaldehyde in phosphate-buffered saline (PBS) for 5 min.

2.4. Preparation of dispersed cell aggregates

Freshly dispersed cell aggregates from SMG and PG of C57BL/6 mice were prepared as described previously (McCall et al., 2013; Odusanwo et al., 2012), with modifications (i.e. trypsin digestion was omitted). The mice were anaesthetized and both SMGs and PGs were removed. The glands were finely minced in dispersion medium, consisting of Dulbecco's modified Eagle's medium (DMEM):Ham's F12 (1:1; Hyclone, Logan, UT, USA) and 0.2 mm CaCl2, 1% w/v bovine serum albumin (BSA), 50 U/ml collagenase (Worthington Biochemical, Freehold, NJ, USA) and 400 U/ml hyaluronidase, at 37 °C for 30 min with aeration (95% air:5% CO2). Cell aggregates in dispersion medium were suspended by pipetting at 20 and 30 min. The dispersed cell aggregates were washed with enzyme-free assay buffer (120 mm NaCl, 4 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgSO4, 1 mm CaCl2, 10 mm glucose, 15 mm N-2-hydroxyethylpiperazine-N′-2-ethane-sulphonic acid (HEPES), pH7.4) containing 1% w/v BSA and filtered through a 0.22 μm nylon mesh. The cells were washed again in DMEM:Ham's F12 (1:1) containing 2.5% v/v fetal bovine serum (FBS; Gibco BRL, Gaithersburg, MD, USA) and the following supplements: 0.1 μm retinoic acid; 80 ng/ml epidermal growth factor; 2 nM triiodothyronine; 5 mm glutamine; 0.4 μg/ml hydrocortisone; 5 μg/ml insulin; 5 μg/ml transferrin; 5 ng/ml sodium selenite; and freshly added 100 μg/ml Normocin™ (InvivoGen, San Diego, CA, USA). The cells were centrifuged at 700 rpm for 5 min at room temperature, then the supernatant was removed and the pellet was resuspended in DMEM:Ham's F12 (1:1) with supplements. Finally, the cells were consecutively filtered through 0.7 μm and 0.4 μm nylon meshes (mechanical dispersion).

2.5. Growth of SMG and PG cells on GFR-MG

Aliquots (100 μl) of GFR-MG (8 mg/ml; 2:1 GFR-MG: DMEM-Ham's F12 (1:1) medium; Becton Dickinson Labware, Franklin Lakes, NJ) were allowed to solidify in a 37 °C incubator for 1 h/well in eight-well chambers mounted on no. 1.5 German borosilicate coverglasses (Nalge Nunc International Corp., Naperville, IL, USA). Then cell clusters (3000/well) were plated on the GFR-MG in DMEM:Ham's F12 (1:1) medium with supplements, as defined above. Differentiated 3D cultures of ‘acinar-like’ spheres were used for assays after incubation at 37°C with 95% air and 5% CO2 for 0–14 days. Time-lapse microscopy of both SMG and PG cells were taken to determine cell migration and proliferation, using a Carl Zeiss Axio Observer inverted microscope.

2.6. Immunofluorescence

SMG and PG cell clusters were stained as previously described (Leigh et al., 2014). Briefly, tissues were fixed in 4% w/v paraformaldehyde for 20 min at room temperature, washed three times with PBS, incubated for 5 min with AlexaFluor 568 conjugated phalloidin F-actin stain (1:400 dilution in PBS; Sigma, St. Louis, MO, USA) and washed three times with PBS. Cell clusters were stained with Propidium Iodide Nucleic Acid Stain (1:3000 dilution in 2× SSC; Invitrogen, Carlsbad, CA, USA) for 5 min. Images were obtained using a Carl Zeiss 510 confocal microscope and analysed using ZEN software (black edition; Carl Zeiss, Thornwood, NY, USA).

2.7. Western blot

Western blot analyses were performed as described previously. Rabbit anti-α-amylase (1:500; Cell Signaling, Danvers, MA, USA) was prepared in 3% w/v BSA in TBST. Peroxidase-linked goat anti-rabbit IgG was used as a secondary antibody (1:5000; Santa Cruz Biotechnology, Santa Cruz, CA, USA). For signal normalization, membranes were treated with stripping buffer (Pierce Biotechnology, Rockford, IL, USA) and reprobed with rabbit β-tubulin (1:500; Cell Signaling), followed by incubation with a peroxidase-linked goat anti-rabbit IgG secondary antibody (1:5000; Santa Cruz Biotechnology). The membranes were treated with Clarity™ chemiluminescence detection reagent (Bio-Rad), and protein bands were visualized and quantified using a ChemiDoc® MP/Image Lab v 4.1 system (Bio-Rad).

2.8. Calcium measurements

Relative intracellular-free Ca2+ concentration was visualized in SMG and PG cell clusters grown on GFR-MG. Cells were preloaded with Fluo-2 AM (TEFLabs, Austin, TX, USA), a Ca2+-sensitive fluorescent dye, for 20 min at 37°C, and washed with assay buffer (136 mm NaCl, 4 mm KCl, 1.2 mm KH2PO4, 1.2 mm MgSO4, 1 mm CaCl2, 10 mm glucose, 15 mm 137 HEPES, pH 7.4, and 0.1% w/v BSA). The release was measured using a Leica DMI6000B fluorescence microscope (Leica Microsystems, Mannheim, Germany) and stimulated with carbachol (100 μm, Sigma) or 1× PBS (negative control) at room temperature. Fluo-2 AM fluorescence images were captured with an ORCA-R2 camera (Hamamatsu Photonics KK, Hamamatsu City, Japan). The graph was obtained using the average pixel intensity values of images taken over time and were normalized to changes in background intensity.

2.9. Electron microscopy

SMG cell clusters were cultured for 2, 7 and 14 days. Then, SMG cell clusters were fixed in 2% v/v glutaraldehyde in 0.1 M cacodylate buffer, pH 7.2, overnight at 4 °C. Cell clusters were rinsed four times for 10 min each with PBS, post-fixed with 1% w/v osmium tetroxide (OsO4) in PBS at pH 7.4 for 10 min each, and rinsed four times for 10 min each with PBS. The samples were then dehydrated with consecutive ethanol washes (25%, 50%, 75%, 95% and 100%) for 10 min each. This was followed by a wash with an equal mixture of ethanol and acetone for 10 min, followed by two acetone washes. Cell clusters were infiltrated with EmBed 812-DER 736 (Electron Microscopy Sciences, Hatfield, PA, USA) epoxy resin. Sections of 70 nm thickness were cut on a Reichert ultra-cut E microtome, and placed on grids. The grids were stained with 5% w/v uranyl acetate for 30 min and Reynold's lead citrate stain for 5 min. The samples were viewed and photographed in a Hitachi SU-70 operating at 30 kV in transmission electron microscopy (TEM) mode.

2.10. Statistical analysis

Data are given as mean ± SEM of results from three or more experiments; p < 0.05 was regarded as significant, calculated using unpaired two-tailed t-tests.

3. Results

3.1. SMG cell clusters form more organized and larger structures than PG cell clusters

As shown in Figure 1, SMG cell clusters were able to form organized hollow single and multilumen structures after 7 and 14 days in culture. In contrast, PG cell clusters were able to form round structures lacking central hollow lumina at 2–14 days. At day 2, we observed comparable SMG and PG cell clusters organized in round islands, lacking a central lumen, with an F-actin ring (Figure 1A–D). At day 7, the SMG cell clusters were highly organized and displayed multiple lumina, with F-actin rings reaching 100–125 μm in diameter (Figure 1B). However, PG cell clusters failed to achieve the degree of organization observed in SMG cell clusters (i.e. lack of an F-actin ring and only 50 μm in diameter; Figure 1E). At day 14, SMG cell clusters maintained acinar organization and developed multiple hollow lumina, reaching 150–250 μm in diameter (Figure 1C). PG cell clusters were unable to develop multiple lumina and failed to display the degree of organization observed in SMG at the same period (Figure 1F). The presence of multiple lumina in SMG acinar-like structures is the result of cell clusters migrating toward each other, attaching and developing an F-actin ring (see supporting information, Movie S1).

3.2. SMG cell clusters maintain α-amylase expression over time

As shown in Figure 2, SMG cell clusters at day 0 displayed equivalent expression of α-amylase, as compared to PG cell clusters. Interestingly, at day 2, SMG clusters displayed higher α-amylase expression as compared to PG cell clusters; however, these differences were not statistically significant. At day 7, both SMG and PG cell clusters displayed α-amylase expression comparable to that of freshly isolated cells, indicating sustained amylase expression for both cell types. At day 14, SMG clusters displayed lower α-amylase expression than PG clusters, although this was not significant.

Figure 2.

Figure 2

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α-Amylase expression in submandibular gland (SMG) and parotid gland (PG) cell clusters, which were grown on growth factor-reduced Matrigel for 0–14 days. After 2–14 days, cells were lysed with Laemmli buffer. Protein extracts were subjected to SDS–PAGE and immunoblotted for α-amylase and β-tubulin. Data are expressed as mean ± SEM of results from three or more experiments

3.3. SMG cell clusters maintain agonist-induced intracellular calcium signalling over time

As shown in Figure 3, SMG cell clusters (days 2–14) were able to respond to the secretory agonist carbachol (100 μm). However, PG cells were only able to respond to carbachol at day 2, while there was an absence of intracellular calcium responses at days 7 and 14.

3.4. SMG cell clusters maintain organelles and cell junctions

SMG cell clusters showed a more defined morphology and responded to secretory agonists for a longer time period than PG cell clusters. Therefore, we studied SMG cell cluster morphology using TEM. At day 2, SMG cells were closely packed and organized in round structures of approximately 30–50 μm diameter, with a lumen-like central space with numerous intercellular canaliculi, i.e. intercellular extensions of the lumen, which occur in salivary gland serous or seromucous acini, but not ducts (Yeh et al., 1991) (Figure 4A). We observed full and empty secretory granules as well as lipid vesicles scattered throughout the cytoplasm, and numerous ribosomes (Figure 4B). In some cases we observed indentations in the luminal membrane that were consistent with sites of secretory granule constitutive exocytosis (Figure 4A). Furthermore, cells were joined along their apical plasma membrane by junctional complexes (Figure 4C). The cytoplasm was rich in mitochondria and dilated profiles of rough endoplasmic reticulum and Golgi apparatus (Figure 4C). The cells also displayed microvilli towards the lumen of the acinar-like structure (Figure 4B, C). At day 7, the cells were organized in layers, forming round structures of approximately 100–150 μm diameter, with lumen-like central spaces (Figure 4D). The varied structures exhibited numerous full secretory granules (Figure 4E, F) with few exocytotic processes (Figure 4D). Similar to what we observed at day 2, cells were joined along their apical plasma membrane by junctional complexes at day 7 (Figure 4F). The cytoplasm also contained numerous mitochondria, ribosomes, scattered rough endoplasmic reticulum and Golgi zones (Figure 4D–F). SMG cell clusters at 14 days still maintained secretory granules (Figure 4G–I); however, some of them were consistent with lipid vesicles (Figure 4H). The acinar-like structures displayed tight junctions and intercellular canaliculi (Figure 4G–I).

Figure 4.

Figure 4

Figure 4

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Localization of secretory granules and cell junctions in mouse SMG cell clusters by TEM. Shown are TEM images of SMG cell clusters grown on growth factor-reduced Matrigel for (A–C) 2 days, (D–F) 7 days and (G–I) 14 days. Cells were processed for morphological analysis as described in Materials and methods. Blue arrows, intercellular canaliculi; red arrows, lipid vesicles; orange arrows, indentations in the luminal membrane that are consistent with sites of exocytosis of secretory granules; green arrows, empty and full secretory granules; yellow arrows, tight junctions (TJ), adherent junctions (AJ) and desmosomes (DM); Mv, microvilli; L, lumen; RER, rough endoplasmic reticulum; M, mitochondria; G, Golgi apparatus

4. Discussion

Our previous study isolated single PG cells using trypsin digestion, followed by two consecutive filtration steps (McCall et al., 2013). We have modified this protocol by removing trypsin digestion, as single cells obtained under these conditions did not respond to carbachol after 3 days in culture (McCall et al., 2013). Using this new approach, we isolated cell clusters from SMGs and PGs, which were capable of forming acinar-like 3D structures (Figure 1). The 3D nature of salivary cells means that they form multilayered spherical structures (Figure 4), which exhibit lumina that are closed off (Figure 1). These structures display secretory machinery; however, we are not yet sure whether secretory products can escape after day 2.

We were able to observe that some lumina developed connectivity, as shown in the z-stack confocal images of SMG acinar-like structures (data not shown). Cell connectivity is important, as it plays a pivotal role in regulating cell survival and differentiation (Giancotti, 1997). The formation of these types of structure could be the initiation of salivary epithelial tubule remodelling. Therefore, the in vitro system presented here would contrast with the classical epithelial branching morphogenesis, which involves cleft formation, end bud expansion and duct elongation (Molnick and Jaskoll, 2000). We observed multilumen connectivity (data not shown), typical of salivary gland branched structures. However, branching morphogenesis does not occur in the SMG cell clusters, but rather an assembly of different cell types. Further studies will be necessary to characterize the extent of cell populations and their function in this cell system.

It is unclear why SMG cells formed more organized structures than PG cells. We believe that different glandular types might play a role. For instance, mouse SMGs possess seromucous acini with secretory granules that contain mucins and small amounts of α-amylase (Amano et al., 2012; Suckow et al., 2001). In contrast, mouse PGs consist of serous acinar cells with secretory granules rich in α-amylase, but lack mucins (Amano et al., 2012; Suckow et al., 2001). Heavily glycosylated high molecular weight glycoproteins, mucins, are involved in the protection and lubrication of luminal epithelial surfaces (Anderson and Fogelson, 1936). Outside of their protective function, transmembrane mucins also engage in signal transduction, through extracellular domain-mediated ligand binding or by interacting with receptors for growth and differentiation factors (Singh and Hollingsworth, 2006). Transmembrane mucins activate signal transduction pathways at the level of small GTPases (Clevers, 2004), which regulate cell polarity (Drees et al., 2001). Based on this information, we believe that the presence of mucins could potentially influence how salivary cells grow in culture.

A difference between rat SMGs and PGs can also be found in their granular ducts, as the former exhibit long cuboidal ducts with prominent secretory granules (in female mice, used in this study) (Denny et al., 1990) and the latter exhibit cuboidal ducts with few secretory granules (Young and Van Lennep, 1978). Previous studies have shown that EGF, HGF and TGFβ are exclusively localized in the secretory granules of granular convoluted tubule cells (Amano and Iseki, 2001). Therefore, it is likely that the high amount of secretory granules could serve as a source for cell growth and differentiation in the cell culture system used here. The myoepithelial cells of mouse SMGs surround both the acini and the intercalated ducts, while in the PGs they surround only the intercalated ducts (Young and Van Lennep, 1978). However, myoepithelial cells seemed to be absent in our cultures (Figure 4).

Note that cell junctions observed in our cultures (Figure 4) might have been in these structures at the time of plating. However, due to the observed cell migration and proliferation (see supporting information, Movie S1), some of these junctions are likely to be formed over the course of the culture. These results are consistent with our previous studies, showing that single cells are able to form tight junctions during culture (McCall et al., 2013).

The acinar-like structures observed here formed secretory canaliculi, typical of seromucous acini (Hand and Oliver, 1977; Parks, 1961). Although the cells maintained secretory structures, the secretory granules at day 14 began to decay in shape. These results indicate a major but incomplete shut-down of the acinar cellular machinery for protein synthesis and storage of secretory granules in rough endoplasmic reticulum. A possible explanation for this shut-down is that granules begin to autophagocytose in the absence of agonist-induced secretion. Alternatively, this might occur because the acinar structures are thick enough that the lumina become closed off to the extracellular environment after day 2. Conceivably, this could be analogous to the ligated duct model, in which the pressure or back-up of secretory products causes a similar outcome (Ahn et al., 2000; Carpenter et al., 2007).

Salivary cells from humans and rodents grown on plastic are known to dedifferentiate (e.g. lose α-amylase expression) after 24 h in culture (Quissell et al., 1994a, 1994b; Yeh et al., 1991). Our recent results indicate that the growth factors EGF and IGF-1, polymerized with fibrin hydrogels, induce α-amylase expression in single PG cells (McCall et al., 2013). In the present study, we observed that SMG cell clusters plated on GFR-MG were able to maintain α-amylase expression (Figure 2). This result indicates the presence of acinar cells in our cell system. Additionally, we could speculate that the isolation method used here is more effective than trypsin digestion, as used previously (McCall et al., 2013). Our laboratory, as well as others, is trying to retain α-amylase expression and tight junction polarity using different approaches. We were able to show that α-amylase expression can be maintained in culture using less harsh culture conditions (Figure 2), which is consistent with the presence of secretory granules (Figure 4). However, we still need to show that α-amylase is active (able to degrade carbohydrates) and that agonists can induce its release from secretory granules. Despite the benefits presented by GFR-MG, its clinical use might be limited due to safety issues (e.g. tumourogenic properties) (Lee et al., 2008; Polykandriotis et al., 2008; Wang et al., 2008). In an effort to retain the benefits of GFR-MG while avoiding associated safety issues, ongoing studies in our laboratory aim to extract the beneficial components of GFR-MG and produce them, independent of their naturally occurring environment. Accordingly, we will determine and dissect the GFR-MG factors responsible for α-amylase expression in acinar cells.

Although the current study was focused on mouse primary cells, we anticipate that similar results could be observed in human salivary glands. Previous studies have shown that the human submandibular gland cell line, HSG, is able to grow on GFR-MG, forming 3D structures capable of expressing the secretory protein histatin-1 (Conti et al., 2011). Human salivary cells cultured on MG also display epithelial tight junctions and secretory proteins (Maria et al., 2011). Interestingly, these cultures did not survive beyond 9 days, therefore the culture method used here could prolong cell survival.

The muscarinic secretory agonist, carbachol, was able to stimulate intracellular calcium mobilization in the acinar-like structures from SMGs (Figure 3). This result indicates that SMG cells are functional for longer periods of time than PG cells. Therefore, SMG cells cultured under the conditions used here are a more amenable model for bioengineering a salivary gland.

In summary, mouse SMG cells were able to respond to a secretory agonist for longer periods of time than PG cells (McCall et al., 2013). We believe that mechanical dispersion followed by filtration (with no trypsin treatment) allows preservation of secretory elements in the acinar-like structures formed in our tissue cultures. This notion is supported by the fact that secretory granules are present at day 14. The acinar-like structures resemble salivary acini, as they display secretory canaliculi and maintain α-amylase expression. Future research will be necessary to fully dissect the presence of different cell types in these clusters (i.e. ductal cells).

Supplementary Material

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Acknowledgments

The authors would like to acknowledge Dr Wade J. Sigurdson, Director of the Confocal Microscopy and Three-dimensional Imaging Core Facility of the School of Medicine and Biomedical Sciences, State University of New York at Buffalo (UB), for assistance in the imaging of specimens; Mr Peter Bush, Director of the South Campus Instrumentation Center of the School of Dental Medicine, UB, for assistance in imaging of specimens; and Dr Robert S. Redman, Oral Pathology Research Laboratory, Department of Veterans Affairs Medical Center, Washington, DC, for critical review of this manuscript. This study was supported by the National Institutes of Health–National Institute of Dental and Craniofacial Research (NIH–NIDCR; Grant Nos 1R01DE021697-01A1 and 1R01DE022971-01, to O.B.).

Footnotes

Conflict of interest: The authors have declared that there is no conflict of interest.

Supporting information: Additional supporting information may be found in the online version of this article at the publisher's web-site.

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