基于干细胞的疗法已经发展成为人类病理再生医学方法的关键组成部分。外源性干细胞移植利用了干细胞自我更新的潜能,区分,受伤部位的家,并充分逃避免疫系统,以保持抗炎细胞因子的释放,趋化因子,和增长因素。许多病理共同的是促炎巨噬细胞在损伤部位的炎症恶化。越来越多的证据表明,间充质基质细胞(MSC)可以影响髓系细胞的免疫表型和功能,以提高治疗效果。当考虑靶向细胞疗法的策略时,了解MSC可以在炎症环境中调节巨噬细胞的表型的程度是令人感兴趣的。迫切需要进行效力测定以阐明体外这些细胞间相互作用,并深入了解归因于MSC的免疫调节和极化能力的潜在作用机制。以及其他具有免疫调节潜力的细胞。然而,反应的复杂性,在细胞表型和特征方面,这些互动的时机,以及细胞接触的程度,使得对这些相互作用的研究具有挑战性。为研究MSCs与巨噬细胞之间的直接相互作用提供研究工具,我们开发了一种在促炎条件下直接共培养MSC与幼稚巨噬细胞的效力测定。使用这种方法,我们证明了巨噬细胞分泌组和表型的变化,可用于评估细胞样品影响细胞微环境的能力。这些结果表明MSC对巨噬细胞的免疫调节作用,同时揭示了关键的细胞因子和表型变化,这些变化可能会影响其作为潜在细胞疗法的功效。主要特征•该方案使用分化为幼稚巨噬细胞的单核细胞,松散粘附,具有相对同质的遗传背景,类似于外周血单核细胞衍生的巨噬细胞。•该方案需要读板器和具有检测六个荧光团的能力的流式细胞仪。•该方案通过向巨噬细胞添加固定数量的新鲜解冻或培养拯救的MSC来提供共培养条件的定量测量。•该方案使用对分泌组和细胞收获的评估来独立地验证巨噬细胞和MSC之间的相互作用的性质。
Stem cell-based therapies have evolved to become a key component of regenerative medicine approaches to human pathologies. Exogenous stem cell transplantation takes advantage of the potential of stem cells to self-renew, differentiate, home to sites of injury, and sufficiently evade the immune system to remain viable for the release of anti-inflammatory cytokines, chemokines, and growth factors. Common to many pathologies is the exacerbation of inflammation at the injury site by proinflammatory macrophages. An increasing body of evidence has demonstrated that mesenchymal stromal cells (
MSCs) can influence the immunophenotype and function of myeloid lineage cells to promote therapeutic effects. Understanding the degree to which
MSCs can modulate the phenotype of macrophages within an inflammatory environment is of interest when considering strategies for targeted cell therapies. There is a critical need for potency assays to elucidate these intercellular interactions in vitro and provide insight into potential mechanisms of action attributable to the immunomodulatory and polarizing capacities of MSCs, as well as other cells with immunomodulatory potential. However, the complexity of the responses, in terms of cell phenotypes and characteristics, timing of these interactions, and the degree to which cell contact is involved, have made the study of these interactions challenging. To provide a research tool to study the direct interactions between
MSCs and macrophages, we developed a potency assay that directly co-cultures
MSCs with naïve macrophages under proinflammatory conditions. Using this assay, we demonstrated changes in the macrophage secretome and phenotype, which can be used to evaluate the abilities of the cell samples to influence the cell microenvironment. These results suggest the immunomodulatory effects of
MSCs on macrophages while revealing key cytokines and phenotypic changes that may inform their efficacy as potential cellular therapies. Key features • The protocol uses monocytes differentiated into naïve macrophages, which are loosely adherent, have a relatively homogeneous genetic background, and resemble peripheral blood mononuclear cells-derived macrophages. • The protocol requires a plate reader and a flow cytometer with the ability to detect six fluorophores. • The protocol provides a quantitative measurement of co-culture conditions by the addition of a fixed number of freshly thawed or culture-rescued MSCs to macrophages. • This protocol uses assessment of the secretome and cell harvest to independently verify the nature of the interactions between macrophages and MSCs.