Factors influencing multinucleated giant cell formation in vitro.

Macrophages fuse together to form multinucleated giant cells (MGC) in granulomas associated with various pathological conditions. Improved in vitro methods are required to better enable investigations of MGC biology and potential contribution to disease. There is a need for standardization of MGC quantification, purification of MGC populations, and characterization of how cell culture variables influence MGC formation. This study examined solutions to address these needs while providing context with other current and alternative methods. Primary mouse bone marrow-derived macrophages were treated with interleukin-4, a cytokine known to induce fusion into MGC. This model was used to systematically assess the influence of cell stimulant timing, cell seeding density, colony stimulating factors, and culture vessel type. Results indicated that MGC formation is greatly impacted by alterations in certain culture variables. An assessment of previously published research showed that these culture conditions varied widely between different laboratories, which may explain inconsistencies in the literature. A particularly novel and unexpected observation was that MGC formation appears to be greatly increased by silicone, which is a component of a chamber slide system commonly used for MGC studies. The most successful quantification method was fluorescent staining with semi-automated morphological evaluation. The most successful enrichment method was microfiltration. Overall, this study takes steps toward standardizing in vitro methods, enhancing replicability, and guiding investigators attempting to culture, quantify, and enrich MGC.