Gray Andrea, Maguire Timothy, Schloss Rene, Yarmush Martin L
Dept. of Biomedical Engineering, Rutgers, The State University of New Jersey, Piscataway, NJ, 08854.
Biotechnol Prog. 2015 Jul-Aug;31(4):1058-70. doi: 10.1002/btpr.2103. Epub 2015 May 28.
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-1β, interferon [IFN]-β, and IFN-γ), individually and in combinations, for the upregulation of MSC PGE2 secretion and attenuation of macrophage secretion of tumor necrosis factor (TNF)-α, 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-1β potently activated monolayer MSCs to enhance PGE2 production and attenuate macrophage TNF-α. Activation by LPS and IL-1β together synergistically increased MSC PGE2, but did not synergistically reduce macrophage TNF-α. MLR and covariate analysis revealed that macrophage TNF-α 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.
治疗性间充质基质细胞（MSC）功能的诱导取决于患病或受伤组织微环境中存在的激活因子。这些功能包括通过分泌分子（如前列腺素E2（PGE2））调节巨噬细胞表型。许多方法旨在优化基于MSC的治疗，包括使用可溶性因子进行预处理以及将细胞固定在生物材料中。然而，由于通常仅并行操纵少数几个因子，因此MSC功能的优化效率通常较低。我们利用析因实验设计来筛选一组6种分子（脂多糖[LPS]、聚肌苷酸-聚胞苷酸[poly（I:C）]、白细胞介素[IL]-6、IL-1β、干扰素[IFN]-β和IFN-γ），单独或组合使用，以通过活化的MSC条件培养基（CM）上调MSC的PGE2分泌并减弱巨噬细胞分泌促炎分子肿瘤坏死因子（TNF）-α。我们使用多变量线性回归（MLR）和协方差分析来确定最佳因子对单层MSC和藻酸盐封装的MSC（eMSC）功能的差异。筛选结果显示，LPS和IL-1β能有效激活单层MSC，以增强PGE2的产生并减弱巨噬细胞TNF-α的分泌。LPS和IL-1β共同激活可协同增加MSC的PGE2，但不能协同降低巨噬细胞TNF-α。MLR和协变量分析表明，巨噬细胞TNF-α强烈依赖于MSC激活因子、PGE2水平和巨噬细胞供体，而不依赖于MSC培养形式（单层与封装）。结果证明了使用统计方法进行高通量细胞分析的可行性和实用性。这种方法可扩展用于开发激活方案，以在移植前最大化MSC和MSC-生物材料的功能，从而改善MSC治疗。
