制备及胸腺器官切片文化中的应用

The overall goal of this procedure is to prepare thymic slices that in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. This method can help answer key questions about T cell development, such as what signals regulate positive and negative selection of developing T cells in situ. The main advantage of this technique is that thymic slices preserve the integrity of the thymic cortex and medulla, and provide a platform to study the development of defined thymocite subsets within an intact thymic microenvironment.

Begin by pinning a euthanized mouse to a dissection board with the ventral side up. Spray the mouse with 70%ethanol then dab away excess ethanol with gauze to prevent it from entering the thoracic cavity and damaging the tissue. Next, lift the skin at the base of the sternum with a pair of forceps and make a cut through the skin.

Extend the cut of the skin upwards to each forelimb. Make an additional cut at the base of the sternum to separate the diaphragm from the rib cage. Then cut each side of the rib cage towards the clavicle and flip the rib cage over towards the head, exposing the thoracic cavity.

The two lobes of the thymus lie on top of the heart. Use forceps and microdissection scissors to remove the connective tissue around the thymic lobes. Then use a pair of fine tipped curved forceps to lift the individual lobes from underneath, or via remaining connective tissue.

Place the thymic lobes in a 15 milliliter conical tube containing PBS and set aside on ice until needed. First, prepare four percent agarose for embedding by dissolving two grams of low melting point agarose in 50 milliliters of sterile PBS and microwaving on a low setting until the agarose has complete dissolved. Cover the flask with aluminum foil and place in a water bath at 55 degrees Celsius until ready for use.

In a tissue culture hood, add 1.5 milliliters of complete RPMI-1640 media to the wells of a six well cell culture plate. Then place a cell culture insert in each well. Set the plate aside in an incubator at 37 degrees Celsius until needed.

Prepare a slushy ice water bath with water and ice in an ice bucket. Transfer the thymic lobes to a tissue wipe soaked with PBS and carefully remove any remaining connective tissue surrounding each thymic lobe using fine tipped forceps. Transfer to a tissue culture dish containing PBS.

After allowing the agarose to cool until the flask is just warm to the touch, pour the cooled four percent agarose into a tissue mold to a height of approximately one centimeter. Next, use a pair of forceps to carefully grasp the thymic lobe by any remaining connective tissue, and roll it gently on a tissue wipe to dry it without damaging the tissue. Ensure that the tissue dries completely, or it will slide out of the agarose during slicing.

Carefully insert the lobe into the agarose and position it at the bottom of the mold, either horizontally to increase the surface area of each slice, or vertically to increase the number of slices. Place the mold in ice water for five to 10 minutes to allow the agarose to solidify. During this time, prepare the vibratome for slicing by mounting the buffer tray, inserting the vibratome blade and assembling the specimen disk.

Place a piece of laboratory tape on the specimen disk. Lastly, sterilize the clean assembled workspace with 70%ethanol. After the agarose has solidified, invert the mold and press gently at its center to release the agarose embedded lobe.

Now use a sharp blade to trim the excess agarose surrounding the lobe, leaving approximately two millimeters of agarose on each side and 0.5 centimeters at the bottom. Add a drop of tissue glue to the piece of tape on the specimen disk and affix the agarose block to the piece of tape on the specimen disk. Multiple blocks can be glued on the tape.

Next, align the vibratome blade with the top of the agarose block, then fill the buffer tray with sterile PBS until the blade and agarose blocks are fully immersed. Set the cutting parameters on the vibratome. Section the agarose embedded tissue to obtain slices with a thickness of 400 to 500 microns.

Use a bent spatula to collect the thymic slices into a tissue culture plate containing sterile PBS as they are cut. Examine the slices under a light microscope at four X magnification. Choose the slices with intact thymic tissue and surrounding agarose.

Next, use a pipette tip to gently slide a thymic slice from the bent spatula onto the cell culture inserts in the six well plate. Each insert can accommodate up to three slices. Maintain the plate at 37 degrees Celsius until needed.

After isolating thymic lobes from congenic mice, as before, use a sterile 15 milliliter tissue grinder filled with five milliliters of sterile PBS containing two percent FBS to dissociate the lobes manually. Transfer the single cell suspension to a 15 milliliter conical tube. Centrifuge the cells at 545 times G for five minutes at four degrees Celsius.

After discarding the supernatant, re-suspend the cells in one milliliter of one X ACK lysis buffer, and incubate at room temperature for three minutes to lyse the red blood cells. Now fill the tube to 15 milliliters with PBS containing two percent FBS. Centrifuge the cells at 545 times g for five minutes at four degrees Celsius.

After re-suspending the cell pellet in 10 milliliters of PBS containing two percent FBS, pass the cells through a 255 micron mesh filter. Count the cells using a hemocytometer. After centrifuging the cells at 545 times g for five minutes at four degrees Celsius, re-suspend the cell pellet in complete RPMI-1640 media at one to three times ten to the sixth cells per 15 microliters, and then proceed to overlay the thymocytes.

Use a pipette to carefully aspirate any PBS surrounding the thymic slices on the insert without damaging the agarose. Without touching the slice with the pipette tip, overlay 15 microliters of the prepared thymocytes onto each thymic slice. Incubate the plate at 37 degrees Celsius for two hours to allow the cells to migrate into the thymic slices.

After two hours, rinse the thymic slices three times with one milliliter of PBS to remove the excess overlaid thymocytes that have not yet migrated into the tissue. Incubate the plate at 37 degrees Celsius until the thymic slices are ready to be harvested, typically one to 72 hours depending on the experiment. Pre-selection MHC class one restricted T cell receptor transgenic thymocytes were overlaid atop thymic slices and the development of mature CD4 positive or CD8 positive single positive T cells was followed by flow cytometry.

When overlaid onto beta 2m knockout thymic slices, the pre-selection OT-1 T cell receptor transgenic thymocytes remain at the double positive stage and do not generate significant CD8 positive single positive T cells. In contrast, when overlaid on wild type thymic slices containing endogenous selecting ligands, the development of CD8 positive T cells at 72 hours demonstrates the ability of this model to support positive selection. To model negative selection to ubiquitous antigen, CTV labeled thymocytes from wild type mice and CFSE labeled thymocytes from OT-1 T cell receptor transgenic RAG-1 knockout mice were mixed one to one and overlaid onto wild type slices in the presence or absence of an ovalbumin peptide.

After 24 hours of incubation, the relative proportions of overlaid cells were analyzed by flow cytometry. This image shows the relative ratios of live CFSE positive OT-1 transgenic cells to live CTV positive wild type cell slices after incubation with or without ovalbumin peptide. A decrease in the relative proportion of OT-1 transgenic cells represents negative selection of OT-1 transgenic cells in the presence of ovalbumin peptide.

After watching this video, you should have a good understanding of how to prepare thymic slices, isolate thymocytes and overlay cells atop the thymic slices in order to analyze their development in situ. Thymic slices can also be used for other applications, such as immunofluorescence and two photon microscopy to determine thymocyte localization, migration, cellular interactions and T cell receptor signaling during thymic selection.