Reprograming Model of Human Monocyte-derived Macrophages for In-vitro Assays

Our research investigated monocyte and macrophage lineage, emphasizing its remarkable plasticity and ability to integrate multiple signal from the environment to shape the effector response during inflammation, tissue repair, and infections. Macrophage are found throughout the body and coordinate the initiation and resolution of the innate and adapted immunity impacting the protected and immune-mediated pathology. Reprogramming macrophage is the most promising feature in this field.

Emerging omics technology have revolutionaried our understanding of macrophage biology, providing insight into their phenotypic diversity, the functional characteristic, their programming potential, and the developmental origin of these cells. The main obstacle and pitfall in discovering macrophage differentiation and polarization is the heterogeneous experimental conditions and protocols across the literature and the lack of consensus in defining the macrophage term. We demonstrate the reprogramming effect of drug and lipid mediator on macrophage polarization and develop a 3D in vitro model of interaction between macrophage and glioblastoma cells.

Additionally, we have shown that platelets license the monocyte-derived macrophage differentiation to N1 phenotype. To begin, place anticoagulated peripheral blood samples in a centrifuge. Spin it to 200G for 15 minutes at room temperature.

Observe the separation of platelet-rich plasma on top and the cellular fraction, including red and white blood cells, below. Dilute the cellular fraction obtained from the first centrifugation with sterile PBS prewarmed to room temperature. Gently homogenize the solution before proceeding with Ficoll-Hypaque density gradient centrifugation.

Transfer 15 milliliters of Ficoll-Hypaque into a sterile 15-milliliter tube. Carefully and slowly add 30 milliliters of diluted blood on top of the Ficoll, ensuring minimal mixing between the phases. Centrifuge the tube containing the Ficoll and diluted blood at 600G for 25 minutes at room temperature.

With a sterile three-milliliter Pasteur pipette, remove some of the yellow top layer. Carefully collect the interface between the yellow layer and the Ficoll layer with a sterile Pasteur pipette. Transfer the collected peripheral blood mononuclear cells, or PBMCs, into a new 15-milliliter tube containing at least three milliliters of sterile PBS.

Top up the tube with sterile PBS to reach a total volume of 12 to 15 milliliters. Then centrifuge the sample at 600G for 10 minutes. Next, spin down the PBMC suspension at 300G for five minutes at 8 to 10 degrees Celsius.

Re-suspend the cells in the desired volume of sterile cold PBS with 2%FBS. Keep the cells at 4 degrees Celsius until the next step. Transfer the desired volume of PBMC suspension into a five-milliliter polystyrene round-bottom tube.

Then add 10 microliters of positive selection cocktail for every 100 microliters of cell suspension. Mix the suspension thoroughly before incubating at room temperature for 15 minutes. Next, pipette 10 microliters of magnetic nanoparticles per 100 microliters of cell suspension.

Vigorously pipette up and down more than five times to ensure uniform suspension of the magnetic nanoparticles. After a 10-minute incubation at room temperature, add PBS containing FBS and EDTA to the suspension to make the total volume to 2.5 milliliters. Gently pipette the cells up and down two to three times.

Insert the tube into the magnet sorter, and let it stand undisturbed for five minutes. Invert the magnet along with the tube in one continuous motion. Keep the magnet and tube inverted for two to three seconds before returning them to an upright position.

Remove the tube from the magnet, and then add 2.5 milliliters of PBS-FBS-EDTA buffer into the tube again. Gently pipette the cell suspension up and down two to three times to mix it. Insert the tube back into the magnet, and set it apart for five minutes.

In one continuous motion, invert the magnet and tube again, discarding the supernatant fraction. Remove the tube from the magnet, and re-suspend the cells in an appropriate volume of PBS-FBS without EDTA. The positively selected cells are now ready for use.

Now add PBS-FBS buffer to the sorted CD14 cells, making the total volume to 12 to 14 milliliters to dilute any remaining EDTA. Centrifuge at 600G for 10 minutes. After discarding the supernatant, re-suspend the positively selected CD14 monocytes in two milliliters of PBS-FBS buffer.

Count the cells using an automatic cell counter. Keep the cells on ice until culture preparation. For culturing, re-suspend the pellet at a concentration of one million cells per milliliter in supplemented RPMI 1640 medium.

Pipette 250 microliters of the prepared cell suspension into each well of a 48-well plate. Add 250 microliters of antibiotic supplemented RPMI medium containing 50 nanograms per milliliter of macrophage colony-stimulating factor into each well. Incubate the cell culture plate in a humidified incubator to initiate macrophage differentiation.

On day four of macrophage differentiation, replace half of the culture medium from each well. Add cytokines or reagents for the desired polarization conditions, and continue culturing for three more days. Harvest the macrophage-derived monocytes on day seven for phenotypic characterization.

M1 macrophages induced by interferon gamma-plus lipopolysaccharide exhibited the highest expression of CD64, an absence of CD206, and low levels of CD163 and MERTK. M2a macrophages induced by interleukin 4 were characterized by increased CD206 expression, and reduced CD64, CD163, and MERTK levels. M2c macrophages induced by interleukin 10 or dexamethasone exhibited increased CD163 and CD14 expression, intermediate levels of CD64, and an increase in MERTK expression.