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In This Article

  • Summary
  • Abstract
  • Introduction
  • Protocol
  • Representative Results
  • Discussion
  • Acknowledgements
  • Materials
  • References
  • Reprints and Permissions

Summary

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.

Abstract

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.

Introduction

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....

Protocol

1. Preparation of vibratome

  1. 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.
  2. Place and secure an additional sheet of laboratory film over the buffer tray to prevent ice from falling in.
  3. 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.......

Representative Results

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.......

Discussion

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.......

Acknowledgements

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.

....

Materials

NameCompanyCatalog NumberComments
Vibratome VT1000SLeica BiosystemsN/AVibratome for sectioning
Double Edge Stainless Steel Razor BladesElectron Microscopy Sciences72000
AgaroseFisher ScientificBP165-25Low-melt
ParafilmSigma-AldrichP6543
Penicillin-Streptomycin-Amphotericin BLonza17-745HPSA
24-well plateCellTreat229124
Dulbecco’s Phosphate Buffered Saline (DPBS)Gibco14190-144
Loctite UltraGel Control SuperglueLoctiteN/APurchased at hardware store
Natural Red Sable Round PaintbrushPrinceton Art & Brush Co7400R-2
Gentamicin SulfateFisher ScientificICN1676045
TransferrinSigma-AldrichT-8158-100mg
L-glutatmineGibco25030-081
Trace ElementsMP BiomedicalsICN1676549
InsulinFisher Scientific12585014
Epidermal Growth FactorCorning354001
HydrocortisoneSigma-AldrichH0888
Retinoic acidFisher ScientificR2625-50MG
Fetal Bovine SerumGibcoA3160602
DMEM/F12 MediaCorning150-90-CV
Millicell Cell Culture InsertMillipore SigmaPICM0125012 mm, 0.4 um pore size for 24 well plate
0.4% Trypan BlueSigma-AldrichT8154
LIVE/DEAD Cell Imaging Kit (488/570)Thermo-FisherR37601Only used LIVE dye component
Anti-Ki-67 AntibodyCell Signaling Technology9129S
Anti-E-cadherin AntibodyCell Signaling Technology3195S
Anti-Cleaved Caspase-3 AntibodyCell Signaling Technology9661L
Anti-SMA AntibodySigma-AldrichC6198
Anti-amylase AntibodySigma-AldrichA8273
Anti-CD31 AntibodyAbcamab28364
Anti-TUBB3 AntibodyCell Signaling Technology5568S
Alexa Fluor 594 Antibody Labeling KitThermo-FisherA20185
Alexa Fluor 594 PhalloidinThermo-FisherA12381
Bovine Serum AlbuminFisher ScientificBP1600
Triton X-100Sigma-Aldrich21568-2500
Paraformaldehyde PrillsFisher Scientific5027632
New England Nuclear Blocking AgentPerkin Elmer2346249No longer sold
DAPICell Signaling Technology4083S
Prolong Gold Antifade Mounting MediaInvitrogenP36934
Leica SPSII Spectral ConfocalLeica BiosystemsN/AFor confocal imaging
Leica DMIL Inverted Phase Contrast MicroscopeLeica BiosystemsN/A
Cobalt-60 Teletherapy InstrumentAtomic Energy of Canada Ltd Theratron-80N/A
Amac Box, ClearThe Container Store60140Agarose block mold

References

  1. Dirix, P., Nuyts, S., Van den Bogaert, W. Radiation-induced xerostomia in patients with head and neck cancer: a literature review. Cancer. 107 (11), 2525-2534 (2006).
  2. Siegel, R. L., Miller, K. D., Jemal, A. Cancer Statistics, 2017.

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