The main goal of this procedure is to implement a comprehensive protocol for the culture and characterization of microphages on different implant surfaces and characterization of the microphages'subtypes. Several methods are employed as a part of this characterization, including qRT-PCR, EISA, and CLSM, to determine the gene expression profiles, secretory proteins, and cell surface markers. The results show the utility and effectiveness of the implemented protocol in polarizing microphages and implant surfaces, and accurately identifying them based on gene expression, secreted proteins, and cell surface markers.
Furthermore, the markers describe revolt consistent and specific expression patterns that can be used to distinguish different subtypes of MDMs. Overall, the study will contribute to the development and design of immunomodulatory materials to improve and promote successful tissue regeneration processes, prevent implant-associated chronic inflammation, and enhance the successful implant integration. To begin, resuspend the isolated human peripheral blood mononuclear cells in 15 milliliters of pre-warmed monocyte attachment medium, and transfer them to a T-75 cell culture flask.
Incubate the cells at 37 degrees Celsius and 5%carbon dioxide for 90 minutes for adhesion. After incubation, discard the supernatant, and wash the cells once with a pre-warmed RPMI 1640 Medium, supplemented with 10%FBS and 1%penicillin and streptomycin by gently tilting the flask to remove non-adherent or loosely-adhered cells. Add 15 milliliters of complete medium containing 10 nanograms per milliliter macrophage colony stimulating factor to the adherent cells.
And incubate for six days to promote differentiation. After differentiation, remove the culture medium from the flask and wash the cells with 10 milliliters of PBS for five minutes. To detach the adherence cells, add 10 milliliters of pre-warmed cell detachment solution and incubate for 30 minutes.
Then, gently tap the flask to release the cells and transfer them to a 50-milliliter tube. To detach the remaining cells, add 10 milliliters of PBS into the flask and scrap the cells. Transfer the detached cells to the 50-milliliter tube.
Centrifuge the detached cell suspension at 300g for 10 minutes, and discard the supernatant before resuspending the cells in five milliliters of pre-warmed complete medium. Using trypan blue staining, count the cells on a hemocytometer to determine cell number and viability. Prepare the cell suspension by adjusting the cell number to 160, 000 cells per one-milliliter of complete medium.
Next, clean the biomaterial discs ultrasonically in 70%ethanol for five minutes, followed by sterilization in 70%ethanol for 30 minutes. After drying the titanium discs, place them in a non-treated 24-well plate and add one milliliter of prepared cell suspension to each well. Incubate the cells for 48 hours at 37 degrees Celsius and 5%carbon dioxide to obtain M0 macrophages.
To obtain M1 polarized macrophages, add interferon gamma and lipopolysaccharide into the wells of a 24-well plate. For M2 polarization, add interleukin-4 and interleukin-13. Incubate the cells for 48 hours at 37 degrees Celsius and 5%carbon dioxide to induce polarization.
After obtaining polarized monocyte-derived macrophages from human peripheral blood mononuclear cells, collect the cell supernatant in a 1.5-milliliter tube. Transfer the titanium disc to a new 24-well plate. Centrifuge the cell supernatant at 300g for five minutes and transfer the supernatant to a new tube.
Measure the cytokines and chemokines at 450 nanometers according to the specific instructions provided by the manufacturer. Calculate the concentration of secreted cytokines or chemokines using the standard curve. Measure the total protein amount using the bicinchoninic acid protein assay kit.
Normalize the concentration of secreted proteins to milligrams of total protein. Wash the polarized macrophages twice in 800 microliters of PBS. Incubate the discs in 400 microliters of fixation buffer for 20 minutes at room temperature.
After removing the fixation buffer, wash the discs three times in 400 microliters of PBS. Now, incubate the discs with 400 microliters of blocking buffer for 30 minutes to block the unspecific binding sites. Discard the blocking buffer and incubate the discs for one hour with primary antibodies diluted in 400 microliters of staining buffer.
After one hour, remove the primary antibodies and wash the discs three times with 400 microliters of wash buffer. Add fluor for labeled secondary antibodies diluted in staining buffer and incubate for one hour at room temperature in the dark. Following incubation, wash the samples three times in the wash buffer for three minutes each.
Add 10 millimolar DRAQ5 in PBS and incubate for 15 minutes in the dark. Then, remove the supernatant and wash the discs once with PBS. Add one drop of mounting medium, and after five minutes, apply the cover glasses and let the samples dry for one hour.
After drying, seal the edges with clear nail polish. To obtain an overview of cells, image the samples on a confocal laser-scanning microscope under 25X magnification. Change the magnification to 63X to determine the structure and localization of surface markers.
Primary monocyte-derived macrophages were successfully polarized into M1 and M2 macrophages on titanium surfaces, with CCR7 more strongly expressed in M1 macrophages and CD209 in M2 macrophages. qRT-PCR analysis showed successful polarization of primary monocyte-derived macrophages on both titanium and cover slip surfaces with high expression of CCR7 and TNF-alpha for M1 and CD209 and CCL13 for M2.High levels of inflammatory TNF-alpha cytokine and CCL13 chemokine confirmed M1 and M2 polarization at the protein level.
