This protocol allows the evaluation of cell to cell interactions between diverse cell types within a tissue, and ex vivo molecular and pharmacological manipulations that would be less feasible in vivo. The main advantage of this technique is the extended period of time these tissue sections could be maintained in culture allowing their evaluation at later time points. This technique will allow a more rapid screening of therapeutic compounds to correct salivary gland damage and will likely reduce the number of mice used for these studies.
We plan to use this technique to understand the salivary gland response to radiotherapy at intermediate time points and to turn genes on and off at specific intervals. The most difficult aspect of this procedure is the tissue sectioning and preventing the loss of tissue sections during the culture and staining steps. Before beginning the procedure disinfect the detachable vibratome components with 70%ethanol, followed by UV sterilization for at least 30 minutes.
Secure an additional sheet of laboratory film over the buffer tray to prevent ice from falling in, and fill the ice chamber with crushed ice. Remove the laboratory film from the buffer tray and fill the buffer tray with 100 milliliters of ice-cold PBS supplemented with 1%penicillin streptomycin ampicillin, or PSA. Then place a stainless steel razor blade into the blade holder and use a screwdriver to facilitate adjustment of the angle of the blade.
After isolating the salivary glands, place the harvested tissue into a sterile 30 millimeter culture dish containing two milliliters of ice-cold PBS supplemented with 1%PSA. Using autoclaved forceps, transfer the glands to the bottom of embedding mold and cover the tissue with liquid 3%low melting point agarose. Using forceps, adjust the tissue to the middle of the block in the appropriate plane, and place the agarose block on ice for 10 minutes.
When the agarose has hardened, carefully run a razor blade around the outer edge of the mold and let the block slide out onto the UV-sterilized laboratory film. Use the razor blade to cut out an agarose box containing the salivary gland, taking care that the plane of section is straight and parallel to the opposite side of the block. Use superglue to attach the block to the cutting surface and obtain 50 or 90 micrometer thick sections on the vibratome at a 0.075 millimeter per second speed at a 100 hertz frequency.
Then use an autoclaved natural hair paintbrush to transfer the sections into individual wells of a 24 well tissue culture dish containing one milliliter of ice-cold PBS per well as they are obtained. When all of the sections have been acquired, use and autoclaved micro spatula to transfer the sections into individual 0.4 micrometer pour size membrane inserts in each well of a 24 well tissue culture plate containing 300 microliters of vibratome culture medium per well. Then place the plate in a 37 degree Celsius and 5%carbon dioxide incubator with humidity, refreshing the medium in the bottom of each well, and adding 40 microliters of medium to the membrane inserts every other day as needed for up to 30 days.
To irradiate the tissues, transfer the sections to the irradiator facility in a covered styrofoam container to avoid fluctuations in temperature and treat the sections with a single five grade dose of radiation. Then return the cultures to a radiation safe tissue culture incubator. For antibody staining of the tissue sections, wash the samples with at least two five minute washes in sterile PBS and fix the sections in 4%paraformaldehyde at four degrees Celsius overnight.
The next morning wash the sections three times with PBS supplemented with 1%bovine serum albumin and 0.1%Triton X-100, or PBT, before permeabilizing the tissues with 0.3%Triton X-100 in PBS for 30 minutes. Wash the sections three times in PBT as demonstrated before blocking any nonspecific binding with blocking agent supplemented with 1%normal goat serum for one hour. Then incubate the sections overnight in the appropriate primary antibody of interest.
Bright field microscope images of primary 2D salivary gland section cultures reveal the presence of viable tissues for up to 30 days of culture under all concentrations of serum supplementation tested. The proliferative capacity of these representative cultured slices was assessed by Ki-67 immunostaining and this marker was observed at all time points evaluated. A low level of Cleaved Caspase-3 positive cells was also observed at all time points with a small increase detected at day 30.
E-cadherin staining was observed in both the submandibular and parotid gland slices throughout the 30 day culture period. Smooth muscle actin-positive cells and cytoskeletal organization were detected at similar levels throughout the culture period. Similarly, functional proteins such as amylase and aquaporin-5 were also detected, albeit at day 14 the staining appeared to be more granular.
In contrast, CD31 and TUBB3 were not consistently expressed throughout the culture period in both glands, suggesting that there is a diversity of tissue constituents maintained for different periods of time throughout the 30 day culture period. Irradiated slices in both glands mimicked similar proliferative apoptotic and cytoskeletal changes observed in vivo. Be careful when moving the sections from the PBS bath in the vibratome to the culture plate and during the staining procedures as the sections are small and delicate.
The section cultures can be treated with most cell line culture methods with the added benefit of being under conditions that are more closely related to in vivo. We anticipate that this will be a powerful technique for other researchers to incorporate into their experiments. The blade on the vibratome is sharp and can be difficult to insert so be weary when handling.
Also trypan blue dye is toxic and carcinogenic so follow all PP guidelines.
