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&#8217;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 &#38; 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. 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 50–90 μm thick salivary gland sections prior to and following exposure to ionizing radiation.

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.

Cannulation of the Mouse Submandibular Salivary Gland via the Wharton&#039;s Duct

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 ...

Collecting Saliva and Measuring Salivary Cortisol and Alpha-amylase in Frail Community Residing Older Adults via Family Caregivers

Salivary measures have emerged in bio-behavioral research that are easy-to-collect, minimally invasive, and relatively inexpensive biologic markers of stress. This article we present the steps for collection and analysis of two salivary assays in research with frail, community residing older adults-salivary cortisol and salivary alpha amylase. The field of salivary bioscience is rapidly advancing and the purpose of this presentation is to provide an update on the developments for investigators interested in integrating these measures into research on aging. Strategies are presented for instructing family caregivers in collecting saliva in the home, and for conducting laboratory analyses of salivary analytes that have demonstrated feasibility, high compliance, and yield quality specimens. The protocol for sample collection includes: (1) consistent use of collection materials; (2) standardized methods that promote adherence and minimize subject burden; and (3) procedures for controlling certain confounding agents. We also provide strategies for laboratory analyses include: (1) saliva handling and processing; (2) salivary cortisol and salivary alpha amylase assay procedures; and (3) analytic considerations.

We demonstrate: (1) procedures for collection of salivary samples in cognitive impaired older adults by family caregivers in the home setting, (2) procedures for measuring stress activity via salivary cortisol and alpha amylase, and (3) representative profiles. Protocols that allow researchers to study stress-linked processes advance our understanding of biological sensitivity and susceptibility.

Salivary measures have emerged in bio-behavioral research  that are easy-to-collect, minimally invasive, and relatively inexpensive  biologic markers of ...

Studying Pancreatic Cancer Stem Cell Characteristics for Developing New Treatment Strategies

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor initiation, metastasis and resistance to radio- and chemotherapy. Here we describe a specific methodology for culturing primary human pancreatic CSCs as tumor spheres in anchorage-independent conditions. Cells are grown in serum-free, non-adherent conditions in order to enrich for CSCs while their more differentiated progenies do not survive and proliferate during the initial phase following seeding of single cells. This assay can be used to estimate the percentage of CSCs present in a population of tumor cells. Both size (which can range from 35 to 250 micrometers) and number of tumor spheres formed represents CSC activity harbored in either bulk populations of cultured cancer cells or freshly harvested and digested tumors 1,2. Using this assay, we recently found that metformin selectively ablates pancreatic CSCs; a finding that was subsequently further corroborated by demonstrating diminished expression of pluripotency-associated genes/surface markers and reduced in vivo tumorigenicity of metformin-treated cells. As the final step for preclinical development we treated mice bearing established tumors with metformin and found significantly prolonged survival. Clinical studies testing the use of metformin in patients with PDAC are currently underway (e.g., NCT01210911, NCT01167738, and NCT01488552). Mechanistically, we found that metformin induces a fatal energy crisis in CSCs by enhancing reactive oxygen species (ROS) production and reducing mitochondrial transmembrane potential. In contrast, non-CSCs were not eliminated by metformin treatment, but rather underwent reversible cell cycle arrest. Therefore, our study serves as a successful example for the potential of in vitro sphere formation as a screening tool to identify compounds that potentially target CSCs, but this technique will require further in vitro and in vivo validation to eliminate false discoveries.

Pancreatic cancer stem cells (CSCs) can be expanded in vitro using the anchorage-independent sphere culture technique, which represents a powerful tool to study CSC biology and can serve as the first step to develop novel CSC-targeting therapies. Here the methodology for expanding, analyzing and targeting of pancreatic CSCs is provided.

Pancreatic ductal adenocarcinoma (PDAC) contains a subset of exclusively tumorigenic cancer stem cells (CSCs) which have been shown to drive tumor ...

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. ...

Generation of Microtumors Using 3D Human Biogel Culture System and Patient-derived Glioblastoma Cells for Kinomic Profiling and Drug Response Testing

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are time consuming, expensive, and labor intensive. To overcome these obstacles, many research groups have turned to spheroid cultures of cancer cells. While useful, tumor spheroids or aggregates do not replicate cell-matrix interactions as found in vivo. As such, three-dimensional (3D) culture approaches utilizing an extracellular matrix scaffold provide a more realistic model system for investigation. Starting from subcutaneous or intracranial xenografts, tumor tissue is dissociated into a single cell suspension akin to cancer stem cell neurospheres. These cells are then embedded into a human-derived extracellular matrix, 3D human biogel, to generate a large number of microtumors. Interestingly, microtumors can be cultured for about a month with high viability and can be used for drug response testing using standard cytotoxicity assays such as 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) and live cell imaging using Calcein-AM. Moreover, they can be analyzed via immunohistochemistry or harvested for molecular profiling, such as array-based high-throughput kinomic profiling, which is detailed here as well. 3D microtumors, thus, represent a versatile high-throughput model system that can more closely replicate in vivo tumor biology than traditional approaches.

Patient-derived xenografts of glioblastoma multiforme can be miniaturized into living microtumors using 3D human biogel culture system. This in vivo-like 3D tumor assay is suitable for drug response testing and molecular profiling, including kinomic analysis.

The use of patient-derived xenografts for modeling cancers has provided important insight into cancer biology and drug responsiveness. However, they are ...

Depletion of Mouse Cells from Human Tumor Xenografts Significantly Improves Downstream Analysis of Target Cells

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic patient tumors in different assays1. Human tumor xenografts represent the gold standard with respect to tumor biology, drug discovery, and metastasis research 2-4. Tumor xenografts can be derived from different types of material like tumor cell lines, tumor tissue from primary patient tumors4 or serially transplanted tumors. When propagated in vivo, xenografted tissue is infiltrated and vascularized by cells of mouse origin. Multiple factors such as the tumor entity, the origin of xenografted material, growth rate and region of transplantation influence the composition and the amount of mouse cells present in tumor xenografts. However, even when these factors are kept constant, the degree of mouse cell contamination is highly variable.
Contaminating mouse cells significantly impair downstream analyses of human tumor xenografts. As mouse fibroblasts show high plating efficacies and proliferation rates, they tend to overgrow cultures of human tumor cells, especially slowly proliferating subpopulations. Mouse cell derived DNA, mRNA, and protein components can bias downstream gene expression analysis, next-generation sequencing, as well as proteome analysis 5. To overcome these limitations, we have developed a fast and easy method to isolate untouched human tumor cells from xenografted tumor tissue. This procedure is based on the comprehensive depletion of cells of mouse origin by combining automated tissue dissociation with the benchtop tissue dissociator and magnetic cell sorting. Here, we demonstrate that human target cells can be can be obtained with purities higher than 96% within less than 20 min independent of the tumor type.

Human tumor xenografts are vascularized and infiltrated by cells of mouse origin during the growth phase in vivo. To circumvent the bias caused by these contaminating cells during downstream analysis, we developed a method allowing for comprehensive depletion of all mouse cells by magnetic cell sorting.

The use of in vitro cell line models for cancer research has been a useful tool. However, it has been shown that these models fail to reliably mimic ...

Radiation Planning Assistant - A Streamlined, Fully Automated Radiotherapy Treatment Planning System

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated arc therapy (VMAT) plans for patients with head/neck cancer and 4-field box plans for patients with cervical cancer. It is a combination of specially developed in-house software that uses an application programming interface to communicate with a commercial radiotherapy treatment planning system. It also interfaces with a commercial secondary dose verification software. The necessary inputs to the system are a Treatment Plan Order, approved by the radiation oncologist, and a simulation computed tomography (CT) image, approved by the radiographer. The RPA then generates a complete radiotherapy treatment plan. For the cervical cancer treatment plans, no additional user intervention is necessary until the plan is complete. For head/neck treatment plans, after the normal tissue and some of the target structures are automatically delineated on the CT image, the radiation oncologist must review the contours, making edits if necessary. They also delineate the gross tumor volume. The RPA then completes the treatment planning process, creating a VMAT plan. Finally, the completed plan must be reviewed by qualified clinical staff.

Radiation therapy is a highly complex cancer treatment that requires multiple specialists to create a treatment plan and provide quality assurance (QA) prior to delivery to a patient. This protocol describes the use of a fully automated system, the Radiation Planning Assistant (RPA), to create high-quality radiation treatment plans.

The Radiation Planning Assistant (RPA) is a system developed for the fully automated creation of radiotherapy treatment plans, including volume-modulated ...

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 ...

Organotypic Slice Cultures as Preclinical Models of Tumor Microenvironment in Primary Pancreatic Cancer and Metastasis

Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing.
This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 &#181;m thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling.
OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

This protocol describes the preparation of organotypic slice cultures (OTSCs). This technique facilitates the ex vivo cultivation of intact multicellular tissue. OTSCs can be used immediately to test for their respective response to drugs in a multicellular environment.