White Andrea, Jenkinson Eric, Anderson Graham
MRC Center for Immune Regulation, University of Birmingham.
J Vis Exp. 2008 Aug 28(18):905. doi: 10.3791/905.
Stromal cells within lymphoid tissues are organized into three-dimensional structures that provide a scaffold that is thought to control the migration and development of haemopoeitic cells. Importantly, the maintenance of this three-dimensional organization appears to be critical for normal stromal cell function, with two-dimensional monolayer cultures often being shown to be capable of supporting only individual fragments of lymphoid tissue function. In the thymus, complex networks of cortical and medullary epithelial cells act as a framework that controls the recruitment, proliferation, differentiation and survival of lymphoid progenitors as they undergo the multi-stage process of intrathymic T-cell development. Understanding the functional role of individual stromal compartments in the thymus is essential in determining how the thymus imposes self/non-self discrimination. Here we describe a technique in which we exploit the plasticity of fetal tissues to re-associate into intact three-dimensional structures in vitro, following their enzymatic disaggregation. The dissociation of fetal thymus lobes into heterogeneous cellular mixtures, followed by their separation into individual cellular components, is then combined with the in vitro re-association of these desired cell types into three-dimensional reaggregate structures at defined ratios, thereby providing an opportunity to investigate particular aspects of T-cell development under defined cellular conditions [corrected].
淋巴组织内的基质细胞被组织成三维结构，这些结构提供了一个支架，据认为该支架可控制造血细胞的迁移和发育。重要的是，这种三维组织结构的维持似乎对正常基质细胞功能至关重要，二维单层培养通常仅能支持淋巴组织功能的个别片段。在胸腺中，皮质和髓质上皮细胞的复杂网络构成一个框架，在淋巴祖细胞经历胸腺内T细胞发育的多阶段过程时，控制其募集、增殖、分化和存活。了解胸腺中各个基质区室的功能作用对于确定胸腺如何进行自我/非自我识别至关重要。在此，我们描述了一种技术，即利用胎儿组织的可塑性，在酶解后使其在体外重新缔合成完整的三维结构。将胎儿胸腺叶解离成异质细胞混合物，然后将其分离成单个细胞成分，再将这些所需细胞类型以特定比例在体外重新缔合成三维再聚集结构，从而提供了一个在特定细胞条件下研究T细胞发育特定方面的机会[已校正] 。
