The overall goal of this in vitro human preclinical model of osteoclastogenesis derived from peripheral blood monocytes, or PBMCs, cultured with breast cancer cell lines is to mimic cancer cell osteoclast interactions. This method can help to answer key questions in the field of bone metastasis about the effects of cancer cell and bone cell interaction within the bone microenvironment. The main advantage of this technique is that it is a fully human pre-clinical model.
Also, this method can provide insight into bone metastasis. It can also be applied to the study of other bone-related pathological mechanisms, such as osteoporosis. Individuals new to this method may struggle because of the variability among healthy donors and in selecting the appropriate PBMC cell density.
Visual demonstration of this method is critical as they need see our PBMC selection and seeding steps require some manual experience before proficiency can be achieved. Begin by diluting at least 20 milliliters of healthy-donor whole blood in EDTA at a one to one ratio in PBS. After mixing thoroughly, split the blood solution into 30 milliliter aliquots in 50 milliliter conical tubes.
Carefully underlay 15 milliliters of lymphocyte separation medium into the bottom of each tube. Separate the cells by density gradient centrifugation. And pull the white mononuclear-cell containing buffy coats into a new 15 milliliter tube.
Wash the harvested cells two times in 20 milliliters of PBS per wash, followed by lysis of the red blood cells with five milliliters of red blood cell lysis buffer for three to five minutes, on ice. Stop the reaction with 20 milliliters of PBS, and collect the white blood cells by centrifugation. Resuspend the pellet and complete alpha M-E-M for counting.
Then, plate the cells at a seven point five times 10 to the five PBMC per square centimeter concentration in each well of a 24 well plate for their incubation at 37 degrees celsius and five percent CO2. After about three hours, remove the supernatant, debris, unattached cells, and unlysed erythrocytes from the cultures. And add fresh medium, supplemented with MCSF.
Fourteen days after seeding, wash the cells two times with PBS, and fix them in four percent paraformaldehyde for 20 minutes at room temperature. Perform trap staining according to the manufacturer's instructions. The osteoclast-like cells will be TRAP positive with at least four nuclei.
Count the osteoclast-like cells manually under the microscope at a 10X magnification. For cancer cell induced osteoclast differentiation when the cancer cells reach ninety-percent confluency, they're detached by trypsinization and seeded onto zero point four micrometer pore inserts at a four times 10 to the three cells per square centimeter concentration in fresh alpha MEM. The next day, place the seeded inserts over undifferentiated one-day plated mononuclear cell cultures in complete alpha M-E-M, and change the medium every two to three days.
Then, 11 days after the start of the co-culture, transfer the inserts into a new plate containing the appropriate reagent for the desired downstream analysis. Breast cancer cells can sustain osteoclastogenesis, as the number of osteoclasts in the wells induced by cancer cells is similar to that obtained in the positive growth-factor control wells. The number of osteoclasts observed in all of the cultures is reduced however, when the cells are treated with an anti-tumor drug.
Interestingly, the surface areas of matured osteoclasts derived from growth factors are larger than those induced by cancer cells. This effect is not observed in anti-tumor drug treated cultures. Once mastered, this technique can be completed in seven eight hours if it is performed properly.
Following this procedure, additional downstream analysis can be performed as gene-expression analysis, Western Blot, immunofluorescence analysis, or ELISA to answer additional questions about cancer-cell/bone-cell interactions or about anti-tumor drug mechanisms. After its development this technique pave the way for researchers in the field of bone metastasis to study the bone microenvironment, in fully human preclinical model. After watching this video you should have a good understanding of how to co-culture monocytes, undergo differentiation with cancer cells.
Don't forget that working with biological samples and drugs can be extremely hazardous and that precautions such as wearing gloves and lab coats should always be taken while performing this procedure.
