人間の女性の生殖器官から樹状細胞の表現型および機能研究の分離

The overall goal of this procedure is to isolate dendritic cells from different anatomical compartments in the human female reproductive tract to evaluate their phenotypical and functional characteristics. This method can help answer key questions regarding tissue-resident dendritic cells in the female genital tract, such as the anatomical compartmentalization of specific subsets and their functional characteristics. The main advantage of this technique is that our tissue digestion protocol does not cleave surface markers, which allows immediate isolation of dendritic cells without overnight incubation or cell activation.

While this method was optimized to isolate dendritic cells from the human female reproductive tract, it can also be adapted to isolate other immune cells or dendritic cells from other tissues. Demonstrating the procedure will be Fiona Barr and Jared Fortier, technicians in our laboratory. Begin this procedure by rinsing the tissue with modified HBSS.

Place the tissue in a 100 by 15 millimeter squared Petri dish, and add approximately three milliliters of a digestion enzyme cocktail to prevent the tissue from drying out. While using forceps to stabilize the tissue, mince it with a scalpel to increase the surface area of the tissue exposed to the digestion enzyme cocktail. Cut the tissue into small pieces.

Add sufficient digestion enzyme cocktail to cover all tissue pieces, and place the lid on the Petri dish. Incubate at 37 degrees Celsius with 5%carbon dioxide on a rotator at approximately 80 rpm for 45 minutes. Ensure that the rotator speed thoroughly mixes the digestion enzyme cocktail without spilling or producing bubbles.

After 45 minutes, visualize the tissue preparation with an inverted light microscope with the 4X and 10X objectives. A successful enzymatic digestion should release epithelial sheets and glands, as shown in this representative image. Perform the separation of single cells in a sterile environment.

First, place a taut 250-micrometer mesh with holder into a 150 by 15 millimeter squared Petri dish, and ensure that the mesh is about 0.5 centimeters above the surface of the Petri dish. Next, wet the mesh with two milliliters of modified HBSS, and transfer the digested tissue onto the mesh. Using the flat surface of a 10-milliliter syringe plunger, gently but firmly grind the digested minced tissue through the mesh.

Once the tissue fragments have been thoroughly dispersed, elevate the mesh and the holder two to three centimeters above the Petri dish, and rinse with three milliliters of modified HBSS to recover additional cells. Transfer the cell suspension to a 50-milliliter conical tube. Place a 20-micrometer mesh with holder about two to three centimeters above a new Petri dish, and wet with two milliliters of modified HBSS.

Pass the cell suspension through the mesh, and rinse with six milliliters of modified HBSS. Collect the mixed cell suspension, and centrifuge at 500 times g for 10 minutes. Aspirate the liquid, and resuspend the pellet in four milliliters of modified HBSS.

Layer the mixed cell suspension over three milliliters of polysucrose solution for density gradient centrifugation. Centrifuge at room temperature at 500 times g for 30 minutes with no brake. Collect the white band from the top of the polysucrose solution, and wash with PBS.

Once the enriched mixed cell suspension has been collected, count the cells using a hemocytometer on a light microscope with the 10X objective. Spin down all the cells in the cell suspension for 10 minutes. Completely aspirate and discard the supernatant.

Then, add dead cell removal beads, and incubate at room temperature for 15 minutes. After 15 minutes, place a 30-micrometer filter on top of the column to retain any remaining tissue debris or cell aggregates. Rinse the filter and column with dead cell removal buffer.

Then, apply the cell suspension, and allow the bead-labeled cells to flow through the magnetic column. Rinse the column with three milliliters of dead cell removal buffer four times. Collect the flow-through containing the live cells.

Following dead cell removal, spin the cells down. Remove the supernatant, and resuspend the cell pellet in magnetic selection buffer, 80 microliters of buffer per one times 10 to the seven cells. For positive selection of dendritic cell populations, add CD1a or CD14 magnetic beads, and incubate at four degrees Celsius for 15 minutes.

Wash the cells with the magnetic selection buffer, spin down, remove the buffer completely, and resuspend the cells in a minimum of 500 microliters of magnetic selection buffer. Attach the column to the magnet, and add a 30-micrometer filter on top of the column to retain any remaining tissue debris or cell aggregates. Rinse the filter and column with magnetic selection buffer.

Apply the cell suspension to the column. Rinse three times with three milliliters of magnetic selection buffer. Transfer the column to a 15-milliliter tube.

Add five milliliters of magnetic selection buffer, and apply the plunger to release the selected cells from the column. To increase the purity, repeat the purification steps with the recovered cells using a new column. Subsequently, assess cell purity by flow cytometry and microscopy, as described in the text protocol.

Prior to starting this assay, naive T-cells are isolated from peripheral blood mononuclear cells using a commercially available kit. Wash the naive T-cells twice with PBS. Resuspend the cells in 500 microliters of PBS.

The rest of the protocol is performed in the dark. While gently vortexing the cells in the biosafety cabinet, add 500 microliters of a 2X solution of cell proliferation dye to the cells. Incubate the cells at 37 degrees Celsius for 10 minutes.

Stop the cell labeling by adding to the cells five milliliters of cell culture medium with 10%human AB serum. Incubate on ice for five minutes, protected from light. Centrifuge the cells at about 500 times g for seven minutes.

Aspirate the medium, and resuspend the cells in five milliliters of cell culture medium with 10%human AB serum. Prior to the last centrifugation, take an aliquot of cells to count. After the third wash, resuspend the cells in cell culture medium with 10%human AB serum at the desired cell concentration.

To start the dendritic cell allogeneic naive T-cell co-culture, plate the isolated dendritic cells and naive T-cells in a 96-well, round-bottom plate at a one to 15 ratio of dendritic cells to T-cells. Centrifuge the plate at about 500 times g for three minutes to ensure the cells are located at the center of the well. Incubate at 37 degrees Celsius for six days.

Monitor the cells by microscopy. After six days, assess cell proliferation by flow cytometry, as described in the text protocol. This graph shows the range of total number of viable cells recovered after tissue processing and dead cell removal in each female reproductive tract compartment, endometrium, endocervix, and ectocervix.

The number of viable dendritic cells recovered per gram of tissue after magnetic bead isolation is shown next. Dendritic morphology of the isolated cells was determined by microscopy following Giemsa staining. Expression of phenotypic markers before and after bead isolation was determined by flow cytometry.

After CD1a-positive and CD14-positive bead isolation, the purity ranged between 85 and 92%An allogeneic stimulation assay was performed to assess dendritic cell functionality. T-cell cluster formation during proliferation was confirmed by proliferation dye staining. The proliferation was then quantified by flow cytometry.

Shown here is the gating strategy to identify different dendritic cells in the mixed cell suspension. Each gated population in multicolor plots is shown in the next panel. Following the gating strategy, specific dendritic cell subsets are identified.

Low cell numbers are expected, as tissue dendritic cells are rare populations. Once mastered, dendritic cell isolation from the tissues can be done in four to five hours if it is performed properly. After watching this video, you should have a good understanding of how to enzymatically digest and process the tissues to generate a single mixed cell suspension and perform magnetic bead selection of dendritic cells for further characterization.