Induction of therapeutic mesenchymal stromal cell (MSC) function is dependent upon activating factors present in diseased or injured tissue microenvironments. These functions include modulation of macrophage phenotype via secreted molecules including prostaglandin E2 (PGE2). Many approaches aim to optimize MSC-based therapies, including preconditioning using soluble factors and cell immobilization in biomaterials. However, optimization of MSC function is usually inefficient as only a few factors are manipulated in parallel. We utilized fractional factorial design of experiments to screen a panel of 6 molecules (lipopolysaccharide [LPS], polyinosinic-polycytidylic acid [poly(I:C)], interleukin [IL]-6, IL-1, interferon [IFN]-, and IFN-), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-, a pro-inflammatory molecule, by activated-MSC conditioned medium (CM). We used multivariable linear regression (MLR) and analysis of covariance to determine differences in functions of optimal factors on monolayer MSCs and alginate-encapsulated MSCs (eMSCs). The screen revealed that LPS and IL-1 potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-. Activation by LPS and IL-1 together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-. MLR and covariate analysis revealed that macrophage TNF- was strongly dependent on the MSC activation factor, PGE2 level, and macrophage donor but not MSC culture format (monolayer versus encapsulated). The results demonstrate the feasibility and utility of using statistical approaches for higher throughput cell analysis. This approach can be extended to develop activation schemes to maximize MSC and MSC-biomaterial functions prior to transplantation to improve MSC therapies. (c) 2015 American Institute of Chemical Engineers Biotechnol. Prog., 31:1058-1070, 2015