STAT3 and phospho-STAT3 were purchased from Cell Signaling Technology

mixed results, suggesting that it is a difficult phenomenon to control within a standard plate culture format. Discussion In this study, we were able to successfully culture and differentiate MPCs for prolonged culture periods within our MBA. This facilitated combinatorial analysis of the impact of small molecule modulators of Wnt signaling upon the osteogenic differentiation of MPCs. In addition to the observations outlined here regarding Wnt signaling, this work opens 518303-20-3 web opportunities for future applications in which the ability to perform cellbased assays with MPCs within such a platform may be used to examine a wide range of microenvironmental conditions and cellular signaling pathways, as well as differentiation of MPCs into a number of lineages. Importantly, these future applications will benefit broadly from the precisely controlled microenvironmental conditions, ease 19774075 of multiplexing, and reduction in reagent usage inherent in such a platform. By optimizing the conditions of the MBA, we were able to validate a combination of culture parameters, in terms of culture chamber height, medium perfusion rate and culture substrate, that resulted in MPCs, which were viable for the 7 day period required for our assay and also able to undergo osteogenic differentiation. Furthermore, we confirmed that cells were evenly distributed throughout the bioreactor and maintained as a homogenous monolayer- both criteria that are vital in applications where image analysis is used to provided an accurate quantitative readout. As a part of this optimisation process, the exchange rate of the culture medium was selected to ensure cell viability whilst providing the least cellular aggregation. Medium flowrate is known to influence several phenomena: fluid shear force, the diffusive/convective balance of supply of nutrients and factors, and removal of metabolic wastes and secreted factors and so the effect of medium flow in our system was considered. Previous 20573509 in vitro studies using 2-D macroscale models have shown that shear stress does have a significant on osteogenesis but have found that it needs to be in the range of 0.10.5 Pa, with a few studies showing stress values as high as 2 Pa are required. These studies correlate well with the shear stress levels that are expected to occur in vivo within bone . In this current work, the medium flowrate and geometry used results in very low shear stress much smaller than values which have been used to influence MSC osteogenic differentiation therefore the impact of shear stress upon the cellular behaviours observed was thought to be minimal compared to the effects of the factor treatments. Further to the parameter optimization, data from the MBA showed a high degree of consistency between both independent MPC donors and runs. This correlation between runs and donors was at levels comparable to previous work with hESCs in MBAs, which had correlation coefficients of 0.660.69 for a single cell line and provided a high degree of confidence in both the quality and reproducibility of the data obtained from the MBA. Data from the MBA screening showed that all three Wnt modulatory compounds influence osteogenic activity and therefore correlate with previous reports that suggest that Wnt signaling has an important role to play in osteogenesis. However, our results were somewhat surprising given that stimulation of the canonical Wnt pathway had inhibitory effects upon osteogenesis, confounded by our observation that inhibiti