This protocol aims to obtain high amounts of unstimulated macrophages that have been derived from bone marrow and have never been exposed to tissue factors or cytokines. The main advantage of this technique is the number of cells acquired from a single animal. With the right procedure, approximately 2 times 10 to the power of 7 macrophages can be obtained.
This method could help researchers from different areas studying macrophages such as cell biology, pathology, immunology, and parasitology, Because cells are always susceptible to contamination, being careful with all sterile steps and using a laminar flow biosafety cabinet are necessary to maintain cell viability. Demonstrating the procedure will be Izabela Aparecida de Souza. A Master degree student from our laboratory.
To begin the procedure on the properly euthanized eight week old C57BL/6 wild type male mouse, soak the mouse with 70%ethanol solution. Using sterile scissors, make a one centimeter incision along the abdomen and remove the skin until the muscle of the hind legs is fully exposed. Then carefully remove the hind legs at the height of the hip without breaking the femur and tibia.
Put the intact hind legs in a conical centrifuge tube containing 70%ethanol solution. Inside a laminar flow biosafety cabinet, transfer the isolated legs from 70%ethanol to sterile PBS. Using forceps and disinfectant wipes, clean the bones by removing all attached muscles and fascia.
Then, use a sterile surgical scalpel blade to cut the bone epiphysis on both ends, exposing the bone marrow. Fill a 20-milliliter syringe with 10 milliliters of sterile PBS supplemented with 2%penicillin streptomycin solution, and connect a 26-gauge needle to it. Hold the bone using anatomical dissection forceps with one hand.
Use the other hand to insert the needle into the bone cavity carefully without crushing the bone. Then, wash out the inside of the bone with PBS and collect the bone marrow in a sterile conical centrifuge tube. Once washed, the bone cavity should appear white.
Centrifuge the collected cells at 300G and four degrees Celsius for 10 minutes. Discard the supernatant and resuspend the cell pellet in one milliliter of DMEM/F1210 medium. Homogenize the suspension and add another nine milliliters of the medium to bring the total volume up to 10 milliliters.
Add one milliliter of the progenitor cell suspension into each of 10 round plastic Petri dishes, measuring 100 milliliters by 20 millimeters. Spread the cells across the plates with a pipette to obtain a uniform distribution. Then, add nine milliliters of DMEM/F1210 supplemented with 20%of L929 cell supernatant to each dish.
Incubate the dishes at 37 degrees Celsius and 5%carbon dioxide. On the third day, add 10 milliliters of DMEM/F1210 medium supplemented with 20%of L929 cell supernatant to each dish. The resulting 20 milliliters of culture medium in each dish is sufficient for cell growth until maturation.
Discard the supernatants from all culture dishes. Wash each culture dish with 10 milliliters of sterile magnesium and calcium free PBS, prewarmed to 37 degrees Celsius, and discard the solution after washing. Add three milliliters of non-enzymatic cell dissociation solution, prewarmed to 37 degrees Celsius to each dish.
Incubate for 10 minutes at 37 degrees Celsius and 5%carbon dioxide. Then, use an inverted microscope to visualize whether the macrophages dissociate from the cell culture dishes. Using a serological pipette wash the dishes with the non-enzymatic cell dissociation solution repeatedly performing circular movements to ensure complete macrophage dissociation.
Collect and combine the solution from all the culture dishes into a 50 milliliter conical centrifuge tube. Once more, add 10 milliliters of sterile, prewarmed PBS to the empty culture dishes. Centrifuge the conical tube containing the combined collected solution at 300G and four degrees Celsius for 10 minutes.
Discard the supernatant and resuspend the pellet with one milliliter of DMEM/F1210. Homogenize the suspension slowly and carefully by pipetting it up and down without damaging the cells. Dilute a 10 microliter aliquot of the macrophage solution by adding 10 microliters of trypan blue to count the cells using a hemocytometer.
At day seven, each dish would produce approximately 2 million macrophages. Exposure to macrophage colony stimulating factor from L929 supernatant gradually transformed the bone marrow progenitors to mature macrophages, displaying typical spread out morphology due to cytoplasmic extensions. On day three, a few immature macrophages have appeared, having a typical round shape morphology with few membrane projections.
Although they transformed along the entire process, seven days were necessary to achieve an adequate number and maturity. The obtained macrophages displayed a homogenous population in terms of size and granularity in the flow cytometry analysis. Furthermore, 100%of the population expressed the characteristic F480 and CD11B surface molecules, forming a single well-defined population of cells.
The phagocytosis capability of the mature and well differentiated macrophages was confirmed by fluorescence microscopy images showing phagocytose Leishmania major parasites inside the macrophages. The legs should be removed from the animal carefully because it is performed in a non-sterile environment and is the most important source of contamination. Legs must not be broken outside of the laminar flow biosafety cabinet.
The bone marrow derived macrophages obtained from this protocol can be used to subsequently perform macrophage in vitro polarization to understand macrophage physiology.
