This work was supported in part by pilot funding provided by University of Arizona Office of Research and Discovery and National Institutes of Health (R01 DE023534) to Kirsten Limesand. The Cancer Biology Training Grant, T32CA009213, provided stipend support for Wen Yu Wong. The authors would like to thank M. Rice for his valuable technical contribution.

1. Preparation of vibratome
<ol>
	<li>Spray detachable components of the vibratome including the buffer tray, blade attachment, agarose block mold, and laboratory film with 70% ethanol, then UV-sterilize for at least 30 min.</li>
	<li>Place and secure an additional sheet of laboratory film over the buffer tray to prevent ice from falling in.</li>
	<li>Fill the ice chamber with crushed ice, remove the laboratory film from the buffer tray, and fill the buffer tray with 100 mL of ice-cold 1x phosphate buffered saline (PBS) solution supplemented with 1% penicillin-streptomycin-ampicillin (PSA).</li>
	<li>Place a stainless steel razor blade into the blade holder. Use the screwdriver to tighten/loosen and change the angle of the blade.</li>
</ol>
2. Preparation of tissue sample in agarose block and sectioning using the vibratome
<ol>
	<li>Autoclave all necessary dissection and sectioning tools including forceps, scissors, and paintbrushes.</li>
	<li>Prepare 3% low melting point agarose in sterile 1x PBS and microwave until the agarose dissolves into solution. Ensure that the solution does not boil over and swirl the bottle periodically to mix. 
	NOTE: Prevent the low-melting agarose from solidifying by keeping it in a warm water bath. The low-melt agarose is fluid at 37 &#176;C and sets at 25 &#176;C. However, the water bath should be below 40 &#176;C in order to pour the agarose around the tissue at physiological temperature.</li>
	<li>Isolate salivary glands and place dissected tissue into 2 mL ice-cold 1x PBS supplemented with 1% PSA in a sterile, 30 mm culture dish.</li>
	<li>Using autoclaved forceps, remove salivary glands from 1x PBS + 1% PSA solution and position at the bottom of an embedding mold. Fill the mold with liquid 3% low melting point agarose to cover the tissue.</li>
	<li>Using the forceps, adjust the tissue to the middle of the block and position the salivary gland in the appropriate plane. The best cross sections for the submandibular and parotid glands are in the vertical plane.</li>
	<li>Place agarose block on ice for 10 min to harden.</li>
	<li>Carefully run a razor blade around the outer edge of the agarose to loosen and let the block slide out onto the UV-sterilized laboratory film from step 1.1.</li>
	<li>Use a razor blade to cut out an agarose box containing the salivary gland. Ensure that the plane of section is straight and parallel to the opposite side of the block (the surface that will be glued down). Since the agarose does not infiltrate the tissue, do not trim off too much agarose around the tissue so the tissue will be well supported.</li>
	<li>Use superglue to attach the block to the cutting surface.</li>
	<li>Section at 50 &#181;m or 90 &#181;m thickness by vibratome at a speed of 0.075 mm/s and frequency of 100 Hz. 
	NOTE: Setting the vibratome at the highest frequency and lowest blade speed provides the most optimal slices.</li>
	<li>Collect sections in a 24-well tissue culture dish containing ~1 mL ice-cold PBS per well using an autoclaved, natural hair paintbrush, placing the brush in 70% ethanol between slice collections to maintain sterility.</li>
</ol>
3. Culturing sections
<ol>
	<li>Autoclave a micro-spatula and forceps.</li>
	<li>Make stock vibratome culture media with or without fetal bovine serum (FBS): DMEM/F12 media supplemented with 1% PSA, 5 &#181;g/mL transferrin, 1.1 &#181;M hydrocortisone, 0.1 &#181;M retinoic acid, 5 &#181;g/mL insulin, 80 ng/mL epidermal growth factor, 5 mM L-glutamine, 50 &#181;g/mL gentamicin sulfate, and 10 &#181;L/mL trace element mixture. Add an appropriate amount of FBS to make 0%, 2.5%, 5.0%, or 10% solutions.</li>
	<li>Prior to sectioning, add 300 &#956;L of pre-warmed media and place a 12 mm diameter, 0.4 &#181;m pore-size membrane insert into each well of a 24-well tissue culture plate. The media should reach the membrane bottom to create a liquid-air interface for culture.</li>
	<li>Gently lay the salivary sections on top of the membrane insert using the micro spatula and culture at 37 &#176;C in humidified 5% CO2 and 95% air atmosphere incubator.
	<ol>
		<li>Add approximately 300 &#956;L primary culture media into the well and a few drops into the membrane inserts (approximately 40 &#956;L) every other day or as needed. Proper culturing showed that the cells are able to survive and be maintained for up to 30 days ex vivo.</li>
	</ol>
	</li>
</ol>
4. Irradiation of salivary gland sections
<ol>
	<li>Treat sections with a single dose of radiation (5 Gy) using cobalt-60 (or equivalent) irradiator.
	<ol>
		<li>Transport sections to irradiator facility using a covered styrofoam container to avoid fluctuations in temperature. In addition, care needs to be taken during transport back to the laboratory incubator to ensure that media does not splash onto culture lid and induce contamination.</li>
		<li>Place the 24-well plate containing salivary gland sections 80 cm from the radiation source, in the center of a 32&#8221; x 32&#8221; radiation field. Radiation dose calculations and corresponding time in irradiator will vary by instrument and cobalt-60 decay.</li>
	</ol>
	</li>
	<li>Continue monitoring and culturing these sections as described in section 3.</li>
</ol>
5. Viability staining
<ol>
	<li>Aspirate old media and wash slices twice with sterile, pre-warmed 1x PBS.</li>
	<li>Stain sections with trypan blue dye.
	<ol>
		<li>Use a micro-spatula to move the slice from the culture to a glass slide.</li>
		<li>Add 0.4% trypan blue solution to the vibratome slice with enough volume to cover the tissue (10&#8211;20 &#181;L).</li>
		<li>Incubate slices at room temperature (RT) in the trypan blue for 1&#8211;2 min for the dye to penetrate the tissue. Nonviable cells will be stained blue, and viable cells will be unstained.</li>
	</ol>
	</li>
	<li>Stain sections with calcein, AM live-cell dye.
	<ol>
		<li>Add enough volume of stain to cover the section in one well of a 24 well-plate (200&#8211;300 &#181;L).</li>
		<li>Incubate slices at RT for 15 min to allow dye penetration.</li>
		<li>Carefully remove the section from the well and place on a glass slide. Mount slices with 1 drop of mounting media.</li>
		<li>Image sections on a fluorescent microscope at excitation/emission wavelengths of 488 nm and 515 nm.</li>
	</ol>
	</li>
</ol>
6. Antibody staining vibratome sections
NOTE: The following provides a general antibody staining protocol specific for Ki-67; however, this protocol can be used with any antibody. All washes are conducted at RT unless otherwise noted.
<ol>
	<li>In the multi-well tissue culture dish, aspirate off media and wash at least 2x with sterile 1x PBS.</li>
	<li>Fix sections using 4% paraformaldehyde (PFA) overnight at 4 &#176;C.</li>
	<li>Aspirate off 4% PFA and wash 3x with PBT [1x PBS, 1% bovine serum albumin (BSA), 0.1% Triton X-100]. 
	NOTE: Sections can be stored in 1x PBS at 4 &#176;C and stained at a later time. Maximum storage time tested in this manuscript was 3 weeks. Longer storage time will need to be optimized by the individual user if that is desired.</li>
	<li>Permeabilize sections using 0.3% Triton X-100 in 1x PBS for 30 min. 
	NOTE: This step can be modified and optimized for specific antibody staining and permeabilization.</li>
	<li>Pipet off permeabilization solution.</li>
	<li>Wash slices 3x for 5 min with 1x PBS, 1% BSA, and 0.1% Triton X-100 (PBT).</li>
	<li>Block the slices with blocking agent with 1% normal goat serum for 1 h.</li>
	<li>Wash the slices 3x for 5 min with PBT.</li>
	<li>Incubate the slices overnight at 4 &#176;C with 500 &#181;L anti-Ki67 rabbit monoclonal antibody diluted in 1% BSA in 1x PBS. 
	NOTE: For phalloidin staining, skip step 6.9 and proceed using the protocol for the specific phalloidin used. For DNA counterstain, skip to step 6.15.</li>
	<li>Aspirate anti-Ki67 rabbit monoclonal antibody.</li>
	<li>Wash the slices 6x for 5 min in 1x PBS.</li>
	<li>Incubate the slices in 500 &#181;L of fluorescently conjugated secondary antibody compatible with the anti-Ki67 antibody diluted in 1% BSA in 1x PBS at RT for 1.5 h covered from light. 
	NOTE: Based on secondary antibody used, incubation time with the secondary antibody can be further optimized by the individual user. In addition, the secondary antibody is light-sensitive. All subsequent washes must be performed in the dark.</li>
	<li>Aspirate secondary antibody.</li>
	<li>Wash slices for 3x for 5 min with 1x PBS.</li>
	<li>Wash slices for 5 min in deionized water.</li>
	<li>Counterstain slices with DAPI (1&#181;g/mL) for 20 min at RT.</li>
	<li>Wash slices (once) for 5 min in deionized water.</li>
	<li>Mount slices with one drop (~40 &#181;L) of mounting media. To avoid excess bubbles, slowly place the coverslip onto the mounting media on the slide, starting with a 45&#176; angle.
	<ol>
		<li>To prevent crushing the thick sections with the coverslip, mount each slice with a spacer. A spacer can be created by placing a rim of vacuum grease in a square around the tissue section.</li>
		<li>When laying the coverslip, the edges of the coverslip can be sealed with vacuum grease. Press down on the edges of the coverslip with your thumb to firmly adhere it onto the slide. Alternatively, seal the coverslip onto the microscope slide using clear nail polish.</li>
	</ol>
	</li>
</ol>
7. Imaging vibratome sections
<ol>
	<li>Image the stained slides within 5 days of staining.</li>
	<li>Obtain optical sections of the stained vibratome slices using a confocal microscope. With a confocal microscope, obtain a z-stack at a defined step size or take individual images depending on the user&#8217;s experimental design. Images can be examined on any computer screen after confocal collection. 
	NOTE: Due to the thickness of the vibratome slices, it is recommended that a confocal microscope or a scope with z-stack capability is used to visualize details within the samples. For the images used in this manuscript, a 63x oil objective was used; however, this can be tailored and further modified by the individual user and the specific scope used. Recommended pixel resolution for each objective and zoom factor with the assumed Nyquist sampling of 2.5 pixels in X and Y for the smallest optically resolvable structure was used. However, some commentators suggest 2.3 pixels, while others suggest 2.8 pixels. Please refer to the Handbook of Biological Confocal Microscopy22 for further details on how to make these calculations.</li>
</ol>

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The authors have nothing to disclose.

Salivary gland research has utilized a number of culture models, including immortalized 2-D cultures, primary 2-D cultures, 3-D salisphere cultures, and 3-D organ cultures from embryonic explants to ascertain questions on underlying biology and physiology. These culture models have yielded insightful information across a diverse array of research questions and will continue to be important tools in salivary research. The limitations of these culture models include modulation of p53 activity during immortalization, transi...

Proper salivary gland function is essential to oral health and is altered following head and neck cancer treatment with radiotherapy1. In 2017, nearly 50,000 new cases of head and neck cancer were reported in the United States2. Due to the tissue-damaging and frequently irreversible effects of radiation therapy on surrounding normal tissues such as salivary glands, patients are often left with severe side effects and diminished quality of life2,3,4. Common complications caused by radiation damage manifests in symptoms such as xerostomia (the subjective feeling of dry mouth), dental caries, impaired ability to chew and swallow, speech impairments, and compromised oral microbiomes2,3,4. These symptoms collectively can lead to malnutrition and impaired survival in affected individuals5. While salivary gland dysfunction in this population has been well-documented, the underlying mechanisms of damage to acinar cells have been disputed, and there is little integration among different animal models6,7.
The current methods of studying salivary gland function and radiation-induced damage include the use of in vivo models, immortalized cell lines, two-dimensional (2-D) primary cell cultures, and three-dimensional (3-D) salisphere cultures8,9,10,11,12. Traditionally, cell culture models from immortalized cell lines and 2-D cultures involve single layered cells cultured on flat surfaces and are valuable for fast, easy, and cost-effective experimentation. However, artificial cell culture conditions can alter the differentiation status and physiological responses of cells exposed to various conditions, and the results often fail to translate to whole organism models14,15. In addition, immortalized cell cultures require modulation of p53 activity, which is critical for the salivary gland response to DNA damage16,17.
3-D salisphere cultures are enriched for stem and progenitor cells at early time points in culture and have been useful for understanding the radiosensitivity of this subset of salivary gland cells9,18. A critical limitation of all these culture models is they are ineffective in visualizing the 3-D structure of the salivary gland, including the extracellular matrix (ECM) and cell-cell interactions over various layers, which are crucial in modulating salivary secretion15. The need for a method that encompasses the behavior of the tissue as a whole but can also be manipulated under laboratory conditions to study the effects of treatment is necessary to further discover the underlying mechanisms of radiation-induced salivary gland dysfunction.
Live tissue sectioning and culture has been documented previously19,20 and is often used to study brain-tissue interactions21. In previous studies, parotid (PAR) salivary gland tissue from mice was sectioned at approximately 50 &#181;m and cultured for up to 48 h, and analysis of viability, cell death, and function was performed thereafter19. Su et al. (2016) expanded on this methodology by culturing human submandibular glands (SMGs) sectioned at 35 &#181;m or 50 &#181;m for 14 days20. The proposed method is an advancement in that it includes both parotid and submandibular salivary glands sectioned at 50 &#181;m and 90 &#181;m and evaluation of the cultures for 30 days. The ability to cut a range of tissue thickness is important in evaluating cell-cell and cell-ECM interactions that are relevant for cellular processes including apical-basolateral polarity and innervation for secretion. Furthermore, the salivary gland slices were irradiated while in culture to determine the feasibility of this culture model to study radiation-induced salivary gland damage.
The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. To determine the maximum time sections that are viable post-dissection, trypan blue staining, live cell staining, and immunohistochemical staining for cell death were performed. Confocal microscopy and immunofluorescent staining were utilized to evaluate specific cell populations, morphological structures, and levels of proliferation. Tissue section cultures were also exposed to ionizing radiation to determine the effects of radiation on various markers in this 3-D model. Induction of cell death, cytoskeletal disruption, loss of differentiation markers, and compensatory proliferation in irradiated ex vivo cultures were compared to previous studies of in vivo models. This methodology provides a means to investigate the role of cell-cell interactions following radiation damage and provides an experimental model to efficiently evaluate the efficacy of therapeutic interventions (gene manipulations or pharmacological agents) that may be less suitable for in vivo models.

<table><tbody><tr><td>Vibratome VT1000S</td><td>Leica Biosystems</td><td>N/A</td><td>Vibratome for sectioning</td></tr><tr><td>Double Edge Stainless Steel Razor Blades</td><td>Electron Microscopy Sciences</td><td>72000</td><td></td></tr><tr><td>Agarose</td><td>Fisher Scientific</td><td>BP165-25</td><td>Low-melt</td></tr><tr><td>Parafilm</td><td>Sigma-Aldrich</td><td>P6543</td><td></td></tr><tr><td>Penicillin-Streptomycin-Amphotericin B</td><td>Lonza</td><td>17-745H</td><td>PSA</td></tr><tr><td>24-well plate</td><td>CellTreat</td><td>229124</td><td></td></tr><tr><td>Dulbecco&amp;rsquo;s Phosphate Buffered Saline (DPBS)</td><td>Gibco</td><td>14190-144</td><td></td></tr><tr><td>Loctite UltraGel Control Superglue</td><td>Loctite</td><td>N/A</td><td>Purchased at hardware store</td></tr><tr><td>Natural Red Sable Round Paintbrush</td><td>Princeton Art &amp;amp; Brush Co</td><td>7400R-2</td><td></td></tr><tr><td>Gentamicin Sulfate</td><td>Fisher Scientific</td><td>ICN1676045</td><td></td></tr><tr><td>Transferrin</td><td>Sigma-Aldrich</td><td>T-8158-100mg</td><td></td></tr><tr><td>L-glutatmine</td><td>Gibco</td><td>25030-081</td><td></td></tr><tr><td>Trace Elements</td><td>MP Biomedicals</td><td>ICN1676549</td><td></td></tr><tr><td>Insulin</td><td>Fisher Scientific</td><td>12585014</td><td></td></tr><tr><td>Epidermal Growth Factor</td><td>Corning</td><td>354001</td><td></td></tr><tr><td>Hydrocortisone</td><td>Sigma-Aldrich</td><td>H0888</td><td></td></tr><tr><td>Retinoic acid</td><td>Fisher Scientific</td><td>R2625-50MG</td><td></td></tr><tr><td>Fetal Bovine Serum</td><td>Gibco</td><td>A3160602</td><td></td></tr><tr><td>DMEM/F12 Media</td><td>Corning</td><td>150-90-CV</td><td></td></tr><tr><td>Millicell Cell Culture Insert</td><td>Millipore Sigma</td><td>PICM01250</td><td>12 mm, 0.4 um pore size for 24 well plate</td></tr><tr><td>0.4% Trypan Blue</td><td>Sigma-Aldrich</td><td>T8154</td><td></td></tr><tr><td>LIVE/DEAD Cell Imaging Kit (488/570)</td><td>Thermo-Fisher</td><td>R37601</td><td>Only used LIVE dye component</td></tr><tr><td>Anti-Ki-67 Antibody</td><td>Cell Signaling Technology</td><td>9129S</td><td></td></tr><tr><td>Anti-E-cadherin Antibody</td><td>Cell Signaling Technology</td><td>3195S</td><td></td></tr><tr><td>Anti-Cleaved Caspase-3 Antibody</td><td>Cell Signaling Technology</td><td>9661L</td><td></td></tr><tr><td>Anti-SMA Antibody</td><td>Sigma-Aldrich</td><td>C6198</td><td></td></tr><tr><td>Anti-amylase Antibody</td><td>Sigma-Aldrich</td><td>A8273</td><td></td></tr><tr><td>Anti-CD31 Antibody</td><td>Abcam</td><td>ab28364</td><td></td></tr><tr><td>Anti-TUBB3 Antibody</td><td>Cell Signaling Technology</td><td>5568S</td><td></td></tr><tr><td>Alexa Fluor 594 Antibody Labeling Kit</td><td>Thermo-Fisher</td><td>A20185</td><td></td></tr><tr><td>Alexa Fluor 594 Phalloidin</td><td>Thermo-Fisher</td><td>A12381</td><td></td></tr><tr><td>Bovine Serum Albumin</td><td>Fisher Scientific</td><td>BP1600</td><td></td></tr><tr><td>Triton X-100</td><td>Sigma-Aldrich</td><td>21568-2500</td><td></td></tr><tr><td>Paraformaldehyde Prills</td><td>Fisher Scientific</td><td>5027632</td><td></td></tr><tr><td>New England Nuclear Blocking Agent</td><td>Perkin Elmer</td><td>2346249</td><td>No longer sold</td></tr><tr><td>DAPI</td><td>Cell Signaling Technology</td><td>4083S</td><td></td></tr><tr><td>Prolong Gold Antifade Mounting Media</td><td>Invitrogen</td><td>P36934</td><td></td></tr><tr><td>Leica SPSII Spectral Confocal</td><td>Leica Biosystems</td><td>N/A</td><td>For confocal imaging</td></tr><tr><td>Leica DMIL Inverted Phase Contrast Microscope</td><td>Leica Biosystems</td><td>N/A</td><td></td></tr><tr><td>Cobalt-60 Teletherapy Instrument</td><td>Atomic Energy of Canada Ltd Theratron-80</td><td>N/A</td><td></td></tr><tr><td>Amac Box, Clear</td><td>The Container Store</td><td>60140</td><td>Agarose block mold</td></tr></tbody></table>

radiation treatment of organotypic cultures from submandibular and parotid salivary glands models key in vivo characteristics

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with radiotherapy. Experimental approaches to understanding mechanisms of salivary gland dysfunction and restoration have focused on in vivo models, which are handicapped by an inability to systematically screen therapeutic candidates and efficiencies in transfection capability to manipulate specific genes. The purpose of this salivary gland organotypic culture protocol is to evaluate maximal time of culture viability and characterize cellular changes following ex vivo radiation treatment. We utilized immunofluorescent staining and confocal microscopy to determine when specific cell populations and markers are present during a 30-day culture period. In addition, cellular markers previously reported in in vivo radiation models are evaluated in cultures that are irradiated ex vivo. Moving forward, this method is an attractive platform for rapid ex vivo assessment of murine and human salivary gland tissue responses to therapeutic agents that improve salivary function.

Primary 2-D cultures are grown in fetal bovine serum (FBS) supplemented media while primary 3-D salisphere culture are typically cultured in serum-free conditions10,11. In addition, the two previous studies utilizing vibratome cultures from salivary glands cultured their sections in 0% or 10% FBS supplemented media19,20. Mouse submandibular slices were sectioned at a thick...

Watch this Scientific Journal Video about Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics at JoVE.com

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Using three-dimensional organotypic cultures to visualize morphology and functional markers of salivary glands may provide novel insights into the mechanisms of tissue damage following radiation. Described here is a protocol to section, culture, irradiate, stain, and image 50&#8211;90 &#956;m thick salivary gland sections prior to and following exposure to ionizing radiation.

Hyposalivation and xerostomia create chronic oral complications that decrease the quality of life in head and neck cancer patients who are treated with ...

radiation-treatment-organotypic-cultures-from-submandibular-parotid

Research

JoVE Journal

Medicine

8.1K Views.  University of Arizona. Dit protocol maakt de evaluatie van cel-cel interacties tussen diverse celtypen binnen een weefsel, en ex vivo moleculaire en farmacologische manipulaties die minder haalbaar zou zijn in vivo. Het belangrijkste voordeel van deze techniek is de langere periode van tijd deze weefselsecties kunnen worden gehandhaafd in de cultuur waardoor hun evaluatie op latere punten. Deze techniek zal een snellere screening van therapeutische verbindingen om speekselklier schade te corrigeren en zal waarschij...

Video: Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton's Duct

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their head and neck regions for cancer treatment. Both groups of patients experience symptoms such as xerostomia (dry mouth), dysphagia 
(impaired chewing and swallowing), severe dental caries, altered taste, oro-pharyngeal infections (candidiasis), mucositis, pain and discomfort.
One innovative approach of regenerative medicine for the treatment of salivary gland hypo-function is speculated in RS Redman, E Mezey 
et al. 2009: stem cells can be directly deposited by cannulation into the gland as a potent method in reviving the functions of the impaired organ. Presumably, 
the migrated foreign stem cells will differentiate into glandular cells to function as part of the host salivary gland. Also, this cannulation technique is an 
expedient and effective delivery method for clinical gene transfer application.
Here we illustrate the steps involved in performing the cannulation procedure on the mouse submandibular salivary gland via the Wharton's duct 
(Fig 1). C3H mice (Charles River, Montreal, QC, Canada) are used for this experiment, which have been kept under clean conventional conditions at 
the McGill University animal resource center. All experiments have been approved by the University Animal Care Committee and were in accordance with the 
guidelines of the Canadian Council on Animal Care.
For this experiment, a trypan blue solution is infused into the gland through the opening of the Wharton's duct using a 
insulin syringe with a 29-gauge needle encased inside a polyethylene tube. Subsequently, the mouse is dissected to show that the infusions migrated 
into the gland successfully.

A protocol for the cannulation of the mouse submandibular salivary gland via the Wharton's duct is described. For this experiment, the trypan blue solution is used as a dyer to demonstrate how this technique effectively delivers infusions into the targeted gland, and to suggest the reliability of this new approach as a potential clinical drug/cell therapy for the regeneration of salivary glands.

Severe salivary gland hypofunction is frequently found in patients with Sj&ouml;gren's syndrome and those who receiving therapeutic 
irradiation in their ...

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There is little knowledge about in situ, in vivo, tumor hypoxia during intracranial development of malignant brain tumors because of lack of efficient means to monitor it in these deep-seated orthotopic tumors. Bioluminescence imaging (BLI), based on the detection of light emitted by living cells expressing a luciferase gene, has been rapidly adopted for cancer research, in particular, to evaluate tumor growth or tumor size changes in response to treatment in preclinical animal studies. Moreover, by expressing a reporter gene under the control of a promoter sequence, the specific gene expression can be monitored non-invasively by BLI. Under hypoxic stress, signaling responses are mediated mainly via the hypoxia inducible factor-1&alpha; (HIF-1&alpha;) to drive transcription of various genes. Therefore, we have used a HIF-1&alpha; reporter construct, 5HRE-ODD-luc, stably transfected into human breast cancer MDA-MB231 cells (MDA-MB231/5HRE-ODD-luc). In vitro HIF-1&alpha; bioluminescence assay is performed by incubating the transfected cells in a hypoxic chamber (0.1% O2) for 24 hr before BLI, while the cells in normoxia (21% O2) serve as a control. Significantly higher photon flux observed for the cells under hypoxia suggests an increased HIF-1&alpha; binding to its promoter (HRE elements), as compared to those in normoxia. Cells are injected directly into the mouse brain to establish a breast cancer brain metastasis model. In vivo bioluminescence imaging of tumor hypoxia dynamics is initiated 2 wks after implantation and repeated once a week. BLI reveals increasing light signals from the brain as the tumor progresses, indicating increased intracranial tumor hypoxia. Histological and immunohistochemical studies are used to confirm the in vivo imaging results. Here, we will introduce approaches of in vitro HIF-1&alpha; bioluminescence assay, surgical establishment of a breast cancer brain metastasis in a nude mouse and application of in vivo bioluminescence imaging to monitor intracranial tumor hypoxia.

In vivo Bioluminescence Imaging of Tumor Hypoxia Dynamics of Breast Cancer Brain Metastasis in a Mouse Model

Bioluminescence imaging of hypoxia inducible factor-1&alpha; activity is applied to monitor intracranial tumor hypoxia development in a breast cancer brain metastasis mouse model.

It is well recognized that tumor hypoxia plays an important role in promoting malignant progression and affecting therapeutic response negatively. There ...

Isolation of Mouse Salivary Gland Stem Cells

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of saliva into the oral cavity. Serous and mucous acinar cells are the protein and mucous producing factories of the gland respectively, and represent the origin of saliva production. Once synthesised, the various enzymatic and other proteinaceous components of saliva are secreted through a series of ductal cells bearing epithelial-type morphology, until the eventual expulsion of the saliva through one major duct into the cavity of the mouth. The composition of saliva is also modified by the ductal cells during this process.
In the manifestation of diseases such as Sj&ouml;gren's syndrome, and in some clinical situations such as radiotherapy treatment for head and neck cancers, saliva production by the glands is dramatically reduced 1,2. The resulting xerostomia, a subjective feeling of dry mouth, affects not only the ability of the patient to swallow and speak, but also encourages the development of dental caries and can be socially debilitating for the sufferer.
The restoration of saliva production in the above-mentioned clinical conditions therefore represents an unmet clinical need, and as such several studies have demonstrated the regenerative capacity of the salivary glands 3-5. Further to the isolation of stem cell-like populations of cells from various tissues within the mouse and human bodies 6-8, we have shown using the described method that stem cells isolated from mouse salivary glands can be used to rescue saliva production in irradiated salivary glands 9,10. This discovery paves the way for the development of stem cell-based therapies for the treatment of xerostomic conditions in humans, and also for the exploration of the salivary gland as a microenvironment containing cells with multipotent self-renewing capabilities.

An optimized protocol for the isolation of stem cells from the mouse salivary gland is described. The method employs enzymatic and mechanical digestion, and permits isolation of salispheres containing cells with characteristics of stem cells.

Mature salivary glands of both human and mouse origin comprise a minimum of five cell types, each of which facilitates the production and excretion of ...

Biology

Retroductal Nanoparticle Injection to the Murine Submandibular Gland

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally delivering bioactive compounds to the salivary glands, greater tissue concentrations can be safely achieved versus systemic administration. Furthermore, off target tissue effects from extra-glandular accumulation of material can be dramatically reduced. In this regard, retroductal injection is a widely used method for investigating both salivary gland biology and pathophysiology. Retroductal administration of growth factors, primary cells, adenoviral vectors, and small molecule drugs has been shown to support gland function in the setting of injury. We have previously shown the efficacy of a retroductally injected nanoparticle-siRNA strategy to maintain gland function following irradiation. Here, a highly effective and reproducible method to administer nanomaterials to the murine submandibular gland through Wharton's duct is detailed (Figure 1). We describe accessing the oral cavity and outline the steps necessary to cannulate Wharton's duct, with further observations serving as quality checks throughout the procedure.

Local drug delivery to the submandibular glands is of interest in understanding salivary gland biology and for the development of novel therapeutics. We present an updated and detailed retroductal injection protocol, designed to improve delivery accuracy and experimental reproducibility. The application presented herein is the delivery of polymeric nanoparticles.

Two common goals of salivary gland therapeutics are prevention and cure of tissue dysfunction following either autoimmune or radiation injury. By locally ...

Bioengineering

Retroductal Submandibular Gland Instillation and Localized Fractionated Irradiation in a Rat Model of Salivary Hypofunction

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. Irreparable cell damage results in a chronic loss of salivary function that impairs basic oral activities, and increases the risk of oral infections and dental caries. Salivary hypofunction and its complications gravely impact a patient's comfort. Current symptomatic management of the condition is ineffective, and newer therapies to assuage the condition are needed. 
Salivary glands are exocrine glands, which expel their secretions into the mouth via excretory ducts. Cannulation of these ducts provides direct access to the glands. Retroductal delivery of a contrast agent to major salivary glands is a routine out-patient procedure for diagnostic imaging. Using a similar procedure, localized treatment of the glands is feasible. However, performing this technique in preclinical studies with small animals poses unique challenges. In this study we describe the technique of retroductal administration in rat submandibular glands, a procedure that was refined in Dr. Bruce Baum's laboratory (NIH)1, and lay out a procedure for local gland irradiation.

Salivary gland hypofunction, a major adverse effect of head and neck radiotherapy diminishes a patient's quality of life. The demonstration of efficacy of new therapies in animal models is a prerequisite before clinical transition. This protocol describes retroductal administration and local irradiation of rat submandibular glands.

Normal tissues that lie within the portals of radiation are inadvertently damaged. Salivary glands are often injured during head and neck radiotherapy. ...

Growth and Characterization of Irradiated Organoids from Mammary Glands

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell monolayers. Although they cannot completely model in vivo complexity, they retain some functionality of the original organ. In cancer models, organoids are commonly used to study tumor cell invasion. This protocol aims to develop and characterize organoids from the normal and irradiated mouse mammary gland tissue to evaluate the radiation response in normal tissues. These organoids can be applied to future in vitro cancer studies to evaluate tumor cell interactions with irradiated organoids. Mammary glands were resected, irradiated to 20 Gy and digested in a collagenase VIII solution. Epithelial organoids were separated via centrifugal differentiation, and 3D organoids were developed in 96-well low-adhesion microplates. Organoids expressed the characteristic epithelial marker cytokeratin 14. Macrophage interaction with the organoids was observed in co-culture experiments. This model may be useful for studying tumor-stromal interactions, infiltration of immune cells, and macrophage polarization within an irradiated microenvironment.

Organoids developed from mouse mammary glands were irradiated and characterized to assess epithelial traits and interactions with immune cells. Irradiated organoids can be used to better evaluate cell-cell interactions that may lead to tumor cell recruitment in irradiated normal tissue.

Organoids derived from the digested tissue are multicellular three-dimensional (3D) constructs that better recapitulate in vivo conditions than cell ...

Isolation of Salivary Epithelial Cells from Human Salivary Glands for In Vitro Growth as Salispheres or Monolayers

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to restore function of glands damaged by radiation therapies or due to pathologies associated with Sj&#246;gren's syndrome. A second goal of researchers is to define the mechanisms by which viruses replicate within glandular tissue where they can then gain access to salivary fluids important for horizontal transmission. These goals highlight the need for a robust and accessible in vitro salivary gland model that can be utilized by researchers interested in the above mentioned as well as related research areas. Here we discuss a simple protocol to isolate epithelial cells from human salivary glands and propagate them in vitro. Our protocol can be further optimized to meet the needs of individual studies. Briefly, salivary tissue is mechanically and enzymatically separated to isolate single cells or small clusters of cells. Selection for epithelial cells occurs by plating onto a basement membrane matrix in the presence of media optimized to promote epithelial cell growth. These resulting cultures can be maintained as three-dimensional clusters, termed "salispheres", or grown as a monolayer on treated plastic tissue culture dishes. This protocol results in the outgrowth of a heterogenous population of mainly epithelial cells that can be propagated for 5-8 passages (15-20 population doublings) before undergoing cellular senescence.

We present a method for isolating and cultivating primary human salivary gland-derived epithelial cells. These cells exhibit gene expression patterns consistent with them being of salivary epithelial origin and can be grown as salispheres on basement membrane matrices derived from Engelbreth-Holm-Swarm tumor cells or as monolayers on treated culture dishes.

The salivary glands are a site of significant interest for researchers interested in multiple aspects of human disease. One goal of researchers is to ...

Immunology and Infection

Genetic Modification and Recombination of Salivary Gland Organ Cultures

Branching morphogenesis occurs during the development of many organs, and the embryonic mouse submandibular gland (SMG) is a classical model for the study of branching morphogenesis. In the developing SMG, this process involves iterative steps of epithelial bud and duct formation, to ultimately give rise to a complex branched network of acini and ducts, which serve to produce and modify/transport the saliva, respectively, into the oral cavity1-3. The epithelial-associated basement membrane and aspects of the mesenchymal compartment, including the mesenchyme cells, growth factors and the extracellular matrix, produced by these cells, are critical to the branching mechanism, although how the cellular and molecular events are coordinated remains poorly understood 4. The study of the molecular mechanisms driving epithelial morphogenesis advances our understanding of developmental mechanisms and provides insight into possible regenerative medicine approaches. Such studies have been hampered due to the lack of effective methods for genetic manipulation of the salivary epithelium. Currently, adenoviral transduction represents the most effective method for targeting epithelial cells in adult glands in vivo5. However, in embryonic explants, dense mesenchyme and the basement membrane surrounding the epithelial cells impedes viral access to the epithelial cells. If the mesenchyme is removed, the epithelium can be transfected using adenoviruses, and epithelial rudiments can resume branching morphogenesis in the presence of Matrigel or laminin-1116,7. Mesenchyme-free epithelial rudiment growth also requires additional supplementation with soluble growth factors and does not fully recapitulate branching morphogenesis as it occurs in intact glands8. Here we describe a technique which facilitates adenoviral transduction of epithelial cells and culture of the transfected epithelium with associated mesenchyme. Following microdissection of the embryonic SMGs, removal of the mesenchyme, and viral infection of the epithelium with a GFP-containing adenovirus, we show that the epithelium spontaneously recombines with uninfected mesenchyme, recapitulating intact SMG glandular structure and branching morphogenesis. The genetically modified epithelial cell population can be easily monitored using standard fluorescence microscopy methods, if fluorescently-tagged adenoviral constructs are used. The tissue recombination method described here is currently the most effective and accessible method for transfection of epithelial cells with a wild-type or mutant vector within a complex 3D tissue construct that does not require generation of transgenic animals.

A technique to genetically manipulate epithelial cells within whole ex vivo cultured embryonic mouse submandibular glands (SMGs) using viral gene transfer is described. This method takes advantage of the innate ability of SMG epithelium and mesenchyme to spontaneously recombine after separation and infection of epithelial rudiments with adenoviral vectors.

Branching  morphogenesis occurs during the development of many organs, and the embryonic  mouse submandibular gland (SMG) is a classical model for the ...

Murine Salivary Functional Assessment via Pilocarpine Stimulation Following Fractionated Radiation

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In these cases, questions remain regarding disease pathogenesis and effective interventions. An optimized technique that allows functional assessment of the salivary glands is invaluable for investigating exocrine gland biology, dysfunction, and therapeutics. Here, we present a step by step approach to performing pilocarpine stimulated saliva secretion, including tracheostomy and the dissection of the three major murine salivary glands. We also detail the appropriate murine head and neck anatomy accessed during these techniques. This approach is scalable, allowing for multiple mice to be processed simultaneously, thus improving the efficiency of the work flow. We aim to improve the reproducibility of these methods, each of which has further applications within the field. In addition to saliva collection, we discuss metrics for quantifying and normalizing functional capacity of these tissues. Representative data are included from submandibular glands with depressed salivary gland function 2 weeks following fractionated radiation (4 doses of 6.85 Gy).

We present a detailed approach to performing saliva collection, including murine tracheostomy and the isolation of three major salivary glands.

Hyposalivation is commonly observed in the autoimmune reaction of Sj&#246;gren's syndrome or following radiation injury to the major salivary glands. In ...

Real-Time Live Imaging of Mouse Submandibular Gland Regeneration

Interrogating Cell-Cell Interactions in the Salivary Gland via Ex Vivo Live Cell Imaging

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal role played by macrophages in driving the regenerative response. However, our understanding of this critical role has primarily relied on static views obtained from fixed tissue biopsies. To overcome this limitation and gain insights into these interactions in real time, this study outlines a comprehensive protocol for culturing salivary gland tissue ex vivo and capturing live images of cell migration.
The protocol involves several key steps: First, mouse submandibular salivary gland tissue is carefully sliced using a vibratome and then cultured at an air-liquid interface. These slices can be intentionally injured, for instance, through exposure to radiation, to induce cellular damage and trigger the regenerative response. To track specific cells of interest, they can be endogenously labeled, such as by utilizing salivary gland tissue collected from genetically modified mice where a particular protein is marked with green fluorescent protein (GFP). Alternatively, fluorescently-conjugated antibodies can be employed to stain cells expressing specific cell surface markers of interest. Once prepared, the salivary gland slices are subjected to live imaging using a high-content confocal imaging system over a duration of 12 h, with images captured at 15 min intervals. The resulting images are then compiled to create a movie, which can subsequently be analyzed to extract valuable cell behavior parameters. This innovative method provides researchers with a powerful tool to investigate and better understand macrophage interactions within the salivary gland following injury, thereby advancing our knowledge of the regenerative processes at play in this dynamic biological context.

Author Spotlight: Studying Macrophage-Epithelial Cell Interactions in Salivary Gland Regeneration After Injury 

Immunofluorescent imaging is constrained by the ability to observe complex, time-dependent biological processes in just a single snapshot in time. This study outlines a live-imaging approach conducted on precision-cut mouse submandibular gland slices. This approach allows for the real-time observation of cell-cell interactions during homeostasis and the processes of regeneration and repair.

Salivary gland regeneration is a complex process involving intricate interactions among various cell types. Recent studies have shed light on the pivotal ...

Radiation Treatment of Organotypic Cultures from Submandibular and Parotid Salivary Glands Models Key In Vivo Characteristics

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