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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.
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.
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 xe....
1. Preparation of vibratome
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.......
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.......
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.
....Name | Company | Catalog Number | Comments |
Vibratome VT1000S | Leica Biosystems | N/A | Vibratome for sectioning |
Double Edge Stainless Steel Razor Blades | Electron Microscopy Sciences | 72000 | |
Agarose | Fisher Scientific | BP165-25 | Low-melt |
Parafilm | Sigma-Aldrich | P6543 | |
Penicillin-Streptomycin-Amphotericin B | Lonza | 17-745H | PSA |
24-well plate | CellTreat | 229124 | |
Dulbecco’s Phosphate Buffered Saline (DPBS) | Gibco | 14190-144 | |
Loctite UltraGel Control Superglue | Loctite | N/A | Purchased at hardware store |
Natural Red Sable Round Paintbrush | Princeton Art & Brush Co | 7400R-2 | |
Gentamicin Sulfate | Fisher Scientific | ICN1676045 | |
Transferrin | Sigma-Aldrich | T-8158-100mg | |
L-glutatmine | Gibco | 25030-081 | |
Trace Elements | MP Biomedicals | ICN1676549 | |
Insulin | Fisher Scientific | 12585014 | |
Epidermal Growth Factor | Corning | 354001 | |
Hydrocortisone | Sigma-Aldrich | H0888 | |
Retinoic acid | Fisher Scientific | R2625-50MG | |
Fetal Bovine Serum | Gibco | A3160602 | |
DMEM/F12 Media | Corning | 150-90-CV | |
Millicell Cell Culture Insert | Millipore Sigma | PICM01250 | 12 mm, 0.4 um pore size for 24 well plate |
0.4% Trypan Blue | Sigma-Aldrich | T8154 | |
LIVE/DEAD Cell Imaging Kit (488/570) | Thermo-Fisher | R37601 | Only used LIVE dye component |
Anti-Ki-67 Antibody | Cell Signaling Technology | 9129S | |
Anti-E-cadherin Antibody | Cell Signaling Technology | 3195S | |
Anti-Cleaved Caspase-3 Antibody | Cell Signaling Technology | 9661L | |
Anti-SMA Antibody | Sigma-Aldrich | C6198 | |
Anti-amylase Antibody | Sigma-Aldrich | A8273 | |
Anti-CD31 Antibody | Abcam | ab28364 | |
Anti-TUBB3 Antibody | Cell Signaling Technology | 5568S | |
Alexa Fluor 594 Antibody Labeling Kit | Thermo-Fisher | A20185 | |
Alexa Fluor 594 Phalloidin | Thermo-Fisher | A12381 | |
Bovine Serum Albumin | Fisher Scientific | BP1600 | |
Triton X-100 | Sigma-Aldrich | 21568-2500 | |
Paraformaldehyde Prills | Fisher Scientific | 5027632 | |
New England Nuclear Blocking Agent | Perkin Elmer | 2346249 | No longer sold |
DAPI | Cell Signaling Technology | 4083S | |
Prolong Gold Antifade Mounting Media | Invitrogen | P36934 | |
Leica SPSII Spectral Confocal | Leica Biosystems | N/A | For confocal imaging |
Leica DMIL Inverted Phase Contrast Microscope | Leica Biosystems | N/A | |
Cobalt-60 Teletherapy Instrument | Atomic Energy of Canada Ltd Theratron-80 | N/A | |
Amac Box, Clear | The Container Store | 60140 | Agarose block mold |
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