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

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 ?-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. Copyright © 2014 John Wiley & Sons, Ltd.

SUBMITTER: Leigh NJ

PROVIDER: S-EPMC4363090 | biostudies-literature | 2017 Mar

REPOSITORIES: biostudies-literature

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

Leigh Noel J NJ Nelson Joel W JW Mellas Rachel E RE McCall Andrew D AD Baker Olga J OJ

Journal of tissue engineering and regenerative medicine 20140904 3

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 form ...[more]

PMID: 25186108

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Project description:PurposeTo analyze baseline CT/MR-based image features of salivary glands to predict radiation-induced xerostomia 3-months after head-and-neck cancer (HNC) radiotherapy.MethodsA retrospective analysis was performed on 266 HNC patients who were treated using radiotherapy at our institution between 2009 and 2018. CT and T1 post-contrast MR images along with NCI-CTCAE xerostomia grade (3-month follow-up) were prospectively collected at our institution. CT and MR images were registered on which parotid/submandibular glands were contoured. Image features were extracted for ipsilateral/contralateral parotid and submandibular glands relative to the location of the primary tumor. Dose-volume-histogram (DVH) parameters were also acquired. Features were pre-selected based on Spearman correlation before modelling by examining the correlation with xerostomia (p < 0.05). A shrinkage regression analysis of the pre-selected features was performed using LASSO. The internal validity of the variable selection was estimated by repeating the entire variable selection procedure using a leave-one-out-cross-validation. The most frequently selected variables were considered in the final model. A generalized linear regression with repeated ten-fold cross-validation was developed to predict radiation-induced xerostomia at 3-months after radiotherapy. This model was tested in an independent dataset (n = 50) of patients who were treated at the same institution in 2017-2018. We compared the prediction performances under eight conditions (DVH-only, CT-only, MR-only, CT + MR, DVH + CT, DVH + CT + MR, Clinical+CT + MR, and Clinical+DVH + CT + MR) using the area under the receiver operating characteristic curve (ROC-AUC).ResultsAmong extracted features, 7 CT, 5 MR, and 2 DVH features were selected. The internal cohort (n = 216) ROC-AUC values for DVH, CT, MR, and Clinical+DVH + CT + MR features were 0.73 ± 0.01, 0.69 ± 0.01, 0.70 ± 0.01, and 0.79 ± 0.01, respectively. The validation cohort (n = 50) ROC-AUC values for DVH, CT, MR, and Clinical+DVH + CT + MR features were 0.63, 0.57, 0.66, and 0.68, respectively. The DVH-ROC was not significantly different than the CT-ROC (p = 0.8) or MR-ROC (p = 0.4). However, the CT + MR-ROC was significantly different than the CT-ROC (p = 0.03), but not the Clinical+DVH + CT + MR model (p = 0.5).ConclusionOur results suggest that baseline CT and MR image features may reflect baseline salivary gland function and potential risk for radiation injury. The integration of baseline image features into prediction models has the potential to improve xerostomia risk stratification with the ultimate goal of truly personalized HNC radiotherapy.

Project description:BackgroundThe applications of multiphoton microscopy for deep tissue imaging in basic and clinical research are ever increasing, supplementing confocal imaging of the surface layers of cells in tissue. However, imaging living tissue is made difficult by the light scattering properties of the tissue, and this is extraordinarily apparent in the mouse mammary gland which contains a stroma filled with fat cells surrounding the ductal epithelium. Whole mount mammary glands stained with Carmine Alum are easily archived for later reference and readily viewed using bright field microscopy to observe branching architecture of the ductal network. Here, we report on the advantages of multiphoton imaging of whole mount mammary glands. Chief among them is that optical sectioning of the terminal end bud (TEB) and ductal epithelium allows the appreciation of abnormalities in structure that are very difficult to ascertain using either bright field imaging of the stained gland or the conventional approach of hematoxylin and eosin staining of fixed and paraffin-embedded sections. A second advantage is the detail afforded by second harmonic generation (SHG) in which collagen fiber orientation and abundance can be observed.MethodsGFP-mouse mammary glands were imaged live or after whole mount preparation using a Zeiss LSM510/META/NLO multiphoton microscope with the purpose of obtaining high resolution images with 3D content, and evaluating any structural alterations induced by whole mount preparation. We describe a simple means for using a commercial confocal/ multiphoton microscope equipped with a Ti-Sapphire laser to simultaneously image Carmine Alum fluorescence and collagen fiber networks by SHG with laser excitation set to 860 nm. Identical terminal end buds (TEBs) were compared before and after fixation, staining, and whole mount preparation and structure of collagen networks and TEB morphologies were determined. Flexibility in excitation and emission filters was explored using the META detector for spectral emission scanning. Backward scattered or reflected SHG (SHG-B) was detected using a conventional confocal detector with maximum aperture and forward scattered or transmitted SHG (SHG-F) detected using a non-descanned detector.ResultsWe show here that the developing mammary gland is encased in a thin but dense layer of collagen fibers. Sparse collagen layers are also interspersed between stromal layers of fat cells surrounding TEBs. At the margins, TEBs approach the outer collagen layer but do not penetrate it. Abnormal mammary glands from an HAI-1 transgenic FVB mouse model were found to contain TEBs with abnormal pockets of cells forming extra lumens and zones of continuous lateral bud formation interspersed with sparse collagen fibers.ConclusionsCollagen fibril arrangement and TEB structure is well preserved during the whole mount procedure and light scattering is reduced dramatically by extracting fat resulting in improved 3D structure, particularly for SHG signals originating from collagen. In addition to providing a bright signal, Carmine Alum stained whole mount slides can be imaged retrospectively such as performed for the HAI-1 mouse gland revealing new aspects of abnormal TEB morphology. These studies demonstrated the intimate contact, but relatively sparse abundance of collagen fibrils adjacent to normal and abnormal TEBS in the developing mammary gland and the ability to obtain these high resolution details subject to the discussed limitations. Our studies demonstrated that the TEB architecture is essentially unchanged after processing.

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

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

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