My name's Jeanette Boudreaux, and today I'd like to take you through the steps to culture mirroring myeloid dendritic cells from bone marrow precursors. The myeloid DC lineage is becoming increasingly appreciated for studies of innate and adaptive immune interplay, as well as being increasingly used as a platform for vaccine development. Today we'll take you through the steps to isolate the precursor cells, put them into culture, and differentiate them into dendritic cells.
Afterwards, I'll show you a flow cytometric analysis of the cells before and after maturation. With the toll-like receptor ligand, the cells proliferate in vitro during the culture period, so expect to see about a five to eightfold increase in the total number of cells compared to the precursors that you played on day zero as well. The procedures I show you today are gonna be demonstrated on an open lab bench, but to maintain sterility, I'd recommend doing your cultures in a biological safety cabinet or a lanar flow hood.
To begin, you'll need a 10 mil syringe with a 25 gauge needle filled with PBS, A pair of curved forceps, a pair of straight forceps, sharp scissors, and a pair of blunt scissors that can be used to clip bone. After euthanizing the donor mouse, prepare the legs for incision by spraying them with 70%ethanol, firmly grasped the mouse's ankle using blunt forceps and used sharp scissors to carefully cut away the skin and muscle tissue of the legs. Begin at the achilles tendon to remove the skin from the posterior portion of the leg, being careful not to damage the bone.
Next, clip away the skin and muscle from the anterior portions of the leg. After this step, the deep muscle tissue of the leg should be exposed. Slide one arm of the curved forceps between the tibia and remaining muscle to break the connective tissue that holds them together and use sharp scissors to cut the tendon that each end freeing the muscle to be removed.
Continue to clean the musculature from the tibia in this way until the bone is exposed and no muscle tissue remains. To clean the femur, grasp the knee joint with the blunt forceps and use the curved forceps to once again, tease away the surrounding muscle tissue. Cut away the remaining musculature with the scissors, being careful not to damage the bone.
Continue to clean away muscle tissue until the head of the femur is clearly exposed within the hip joint. Slide the bone scissors along the femur and over the greater trochanter into the hip joint. With one firm cut, separate the bone and place it into PBS on ice.
If many dendritic cells are required, the second leg may also be harvested. To remove the bone marrow, ensure that there's no muscle tissue remaining at the extremities of the bone. Then using the bone scissors, separate the bones of the knee joint.
Then cut off the epiphyses of the first bone. Insert the tip of the needle into the center of the bone, and push firmly on the syringe plunger to force the bone marrow into a clean dish. Continue until you can see that the marrow has been completely flushed, and repeat this with the remaining bones.
Using the rubber end of a plunger from a one mil syringe, gently dissociate the bone marrow into a single cell suspension by mashing with an up and down motion. Collect the bone marrow into a conical centrifuge tube. Rinse the dish with PBS and add this to the tube.
PT the cells by centrifugation for five minutes at 1500 RPM. At this point, some researchers will include a step to lyce red blood cells. However, we have found that this step is unnecessary.
Re suspend the pellet in DC media and count on a hemo cytometer. This preparation is a mixture of bone marrow cells and a fraction are capable of differentiating into dendritic cells. The provision of G-M-C-S-F will favor the development of dcs, but not the other cell types.
Therefore, it is unnecessary to deplete the other cells. For plating, we must count the bone marrow cells, but not the erythrocytes. These are the biggest and brightest cells.
For example, in this field, we would count these cells but not these ones. It's time to plate the cells. For DC culture, we do not use culture coated plates.
Dcs differentiate best in polystyrene. Petri plates plate the cells at a density of 200, 000 precursors per mil in DC media, supplemented with 40 nanograms per mil G-M-C-S-F and incubated 37 degrees for one week. On the third day of culture, refresh the media by adding in half of the total volume of fresh G-M-C-S-F supplemented DC media.
On the sixth day. Refresh the media by replacing one third of it again with G-M-C-S-F containing media to harvest the cells. On day seven, remove the media which contains non-adherent dcs and collect in a conical tube.
Scrape the dish using a tissue culture scraper, then rinse the scraper and dish and add this rinse to the non-adherent cells in the conical tube. At this point, you may begin your experiments. Your dcs are ready.
This is what the cells look like on day one of culture. Just after plating. Note that the cells are small, round, and non-adherent.
After seven days of culture, many but not all of the cells have adhered to the dish. Several of the cells have developed obvious dendrites under these culture conditions. Typically, 70 to 90%of harvested cells are CD 11 C positive.
At this point, the cells are differentiated, but relatively immature. Researchers often choose to promote a mature phenotype by stimulating cells overnight with a toll-like receptor ligand, for example. Here are some representative flow cytometry plots of dcs following overnight stimulation with CPG Phenotypical.
Markers of maturation are shown in the top. Panel CD 40 and CD 86 are upregulated as demonstrated by a shift of the blue line, which represents the stimulated population. Similarly, a dot plot depicting CD 11 C and IL 12 in the lower panel demonstrates production of the cytokine.
In response to CPG, several modifications can now be applied to the dendritic cells. For example, they can be stimulated with tolike receptor ligands or cytokines to improve their maturation status and antigen presenting capacity. Additionally, many vaccine studies have applied peptide or protein pulsing to the dcs prior to their use in vivo as an immunization strategy.
Best of luck with your experiments.
