Animals

Female C57BL/6J mice 6-weeks-old, weighing ~17–20 g, were purchased from the Jackson Laboratory (Bar Harbor, ME). All animal usage, anesthesia, and surgeries were conducted with the approval of the University of Utah Institutional Animal Care and Use Committee, in accordance with their strict guidelines.

Fresh SMG cell isolation

Mice were euthanized using carbon dioxide (CO 2 ) followed by abdominal exsanguination. SMG were then removed, cut into small pieces and placed in a 35 ml GentleMACS™ C Tube containing 6.5% tumor dissociation enzyme mixture (Miltenyi Biotec Inc. Auburn, CA) in DMEM/F12 (Invitrogen, Carlsbad, CA). Subsequently, the tissue was dissociated using a GentleMACS (Miltenyi Biotec Inc.) and incubated in a shaking water bath at 37 °C for 30 min. After three such steps and two intervening incubations, SMG cells were centrifuged at 150 × g for 5 min at 4 °C and the dispersion medium was removed. The cells were then resuspended in 5 ml DMEM/F12 complete medium containing the following: 2.5% FBS, 2 nM triiodothyronine, 0.1 μM retinoic acid, 0.4 μg/ml hydrocortisone, 80 ng/ml EGF, 5 ng/ml sodium selenite, 5 mM glutamine, 5 μg/ml insulin and 5 μg/ml transferrin. Cells were then passed through 70 and 40 µm strainers (Thermo Fisher Scientific, Waltham, MA) and seeded at 1.0 × 106 cells/plate on FBS coated 35-mm UpCell™ temperature-responsive dishes (Thermo Fisher Scientific), cultured at 37 °C in a humidified atmosphere of 95% air-5% CO 2 and used at confluence (a time when monolayer completely covers the plate), with the cell culture medium replaced every other day.

Cell sheet preparation

After 8 days of incubation, temperature was reduced below 25 °C for 30 min to allow the cell sheet to detach from the dish surface. After removing culture medium from single layer cell sheet, a wet transfer membrane was placed over the SMG monolayer and 20 µl of fresh medium was added to prevent the cells from drying out. After 30 min of incubation at room temperature, the attached cell layer was transferred to a new FBS coated culture dish and incubated at 37 °C for 30 min. Then, one milliliter of fresh medium was added on top of the membrane and gently removed from the cell layer. After aspirating the culture medium, the apical side of the first cell sheet layer was covered with the basolateral side of the second cell sheet and incubated at 37 °C for 30 min. Finally, the wet transfer membrane was gently withdrawn from the cell layers and a double layer cell sheet was cultured for one additional day for further experiments.

Intracellular free calcium levels

The intracellular free calcium concentration in both single and double layer cell sheets was measured using a Leica DMI6000B imaging system. Briefly, cell sheets were transferred into a Lab Tek Chamber Slide (Thermo Fisher Scientific). After 1 day of incubation, they were treated with Fura-2-acetoxymethylester (Fura-2 AM, 4 μM) for 30 min at 37 °C in DMEM/F12 and washed two times with DMEM/F12 and two times with assay buffer (DMEM/F12 containing 10 mM glucose, 1 mM CaCl 2 , 0.1% BSA). Then, cells were stimulated with carbachol (Cch, 100 μM). Subsequently, images were recorded and analyzed using a Leica Suite X software.

Animal model

The wounded SMG model was created following a method reported previously.18,19 Briefly, C57BL/6J mice were anesthetized with 3% isoflurane with an oxygen flow rate set at 2.0 L/min, SMG exposed and surgical wounds created using a 3 mm diameter biopsy punch in both glands and treated with a single or double layer cell sheet (experimental group), left untreated (untreated wounded controls) or were left unwounded (sham surgery controls). Then, the skin incision was sutured and post-surgical studies at day 8 were performed. For these purposes, SMG were dissected and processed for histological analysis and saliva secretion studies as described below.

Hematoxylin and eosin staining

Cell sheets were fixed in 4% PFA for 10 min, dehydrated in 70% ethanol solution, embedded in paraffin wax, and cut into 3 μm sections. Then, they were deparaffinized with xylene and rehydrated with serial ethanol solutions and distilled water. Finally, hematoxylin and eosin staining was performed, and specimens were examined using a Leica DMI6000B inverted microscope (Leica Microsystems, Wetzlar, Germany).

Saliva flow rate measurements

To collect stimulated saliva secretion, mice were anesthetized with ketamine (100 mg/kg) and xylazine (5 mg/kg), and injected with pilocarpine (50 mg/kg) and isoproterenol (0.5 mg/kg) via intraperitoneal injection.74,75 Then, stimulated saliva was collected using a micropipette for 5 min.19 Finally, statistical significance was assessed by one-way ANOVA (P < 0.01) and Dunnett’s post hoc test for multiple comparisons to the untreated group and sham control group.

Weight change

Mice were weighed at the start of each experiment and data was collected for 8 days. Then, statistical significance was assessed by two-way ANOVA (P < 0.01) and Dunnett’s post hoc test for multiple comparisons to the untreated group.

Transmission electron microscopy

Cells sheets or tissues were fixed overnight at 4 °C in a solution containing 2.5% glutaraldehyde, 1% paraformaldehyde, 100 mM cacodylate buffer at pH 7.4, 6 mM CaCl 2 , and 4.8% sucrose. The next day, cells were washed three times for 5 min each with cacodylate buffer, post-fixed with 2% osmium tetroxide at room temperature for 45 min, washed twice for 5 min with cacodylate buffer, then washed once with distilled water for 5 min. Specimens were then stained with saturated uranyl acetate for 45 min at room temperature, washed three times for 5 min each with distilled water, then dehydrated with consecutive ethanol washes (30%, 50%, 70%, twice at 95%, and three times with 100%) for 15 min each. This was followed by dehydration with absolute acetone three times for 10 min each. Specimens were infiltrated with consecutive EPON epoxy resin incubations (30% for 5 h, 70% overnight, three times with 100% for 8 h). 70 nm thick sections were made using a Leica Ultra Cut 6 ultratome, and imaged using a JEOL JEM-2800 operated at an accelerating voltage of 200 kV.

Confocal analysis

A detailed procedure of deparaffinization and antigen retrieval methods of 3 μm thick paraffin embedded samples can be obtained from a previous study.18 Specimens were then blocked in 5% goat serum in PBS for 1 h at room temperature, and incubated at 4 °C with the primary antibodies in 5% goat serum overnight as follows: rabbit anti-ZO-1 (Invitrogen, 1:50 dilution), mouse anti-E-cadherin (BD Biosciences, San Jose, CA, 1:100 dilution), rabbit anti-aquaporin 5 (Abcam, Cambridge, MA 1:200 dilution), mouse anti-cytokeratin 7 (Abcam, 1:250 dilution), rabbit anti-TMEM16A (Abcam, 1:50 dilution) and mouse anti-Na+/K+-ATPase α antibody (Santa Cruz Biotechnology, Santa Cruz, CA1:100 dilution). Then, sections were incubated for 2 h with anti-rabbit Alexa Fluor 488 and anti-mouse Alexa Fluor 568 secondary antibody solution at 1:200 dilutions in 5% goat serum at room temperature. Subsequently, specimens were counter-stained with TO-PRO-3 Iodide nuclear stain (Invitrogen) at room temperature for 15 min at 1:1000 dilutions. Finally, specimens were analyzed using a confocal Zeiss LSM 700 microscope (Carl Zeiss, Oberkochen, Germany) at ×20 magnifications for in vivo studies and ×40 for in vitro studies. A total depth of 3 μm was acquired for each sample, and a total projection was visualized in the xy planes.

Reporting summary

Further information on research design is available in the Nature Research Reporting Summary linked to this article.