本视频展示了经2-dGuo处理的胸腺再聚集培养物的制备过程。
本文内容
勘误通知
Important: There has been an erratum issued for this article. View Erratum Notice
摘要
摘要
淋巴组织内的基质细胞以三维结构排列，形成一种被认为可调控造血细胞迁移与发育的支架。值得注意的是，维持这种三维结构对基质细胞的正常功能至关重要，而二维单层培养通常仅能支持淋巴组织部分功能的实现。在胸腺中，皮质与髓质上皮细胞构成复杂的网络结构，作为调控淋巴样前体细胞招募、增殖、分化和存活的框架，这些前体细胞在胸腺内经历多阶段的T细胞发育过程。明确胸腺中各个基质区室的功能作用，对于理解胸腺如何建立自我/非我识别机制至关重要。本文介绍一种利用胎儿组织可塑性使其重新组装为完整三维结构的技术。 体外，经过酶解离后。将胎儿胸腺小叶解离为异质性细胞混合物，随后分离成单独的细胞组分，再与 体外 将这些目标细胞类型以特定比例重新组合成三维再聚集结构，从而可在明确的细胞条件下研究T细胞发育的特定方面。（本文基于首次发表于《分子生物学方法》2007年，第380卷，第185–196页的研究工作。）
方案
有关制备再聚集胸腺培养物的更多信息，请访问 Springer Protocols。
披露
作者无任何利益冲突需要披露。
重印与许可
勘误
Formal Correction: Erratum: Reaggregate Thymus Cultures
Posted by JoVE Editors on 4/01/2012. Citeable Link.
A correction was made to: Reaggregate Thymus Cultures. A revised abstract was republished due to a publisher error. The abstract was corrected to:
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. (This article is based on work first reported Methods in Molecular Biology 2007, Vol. 380 pages 185-196).
from
In the thymus, immature CD4+8+ thymocytes expressing randomly rearranged T-cell receptor α- and b-chain genes undergo positive and negative selection events based on their ability to recognize self-peptide/major histocompatibility complex (MHC) molecules expressed by thymic stromal cells. In vivo analysis of the role of thymic stromal cells during intrathymic selection is made difficult by the cellular complexity of the thymic microenvironment in the steady-state adult thymus, and by the lack of appropriate targeting strategies to manipulate gene expression in particular thymic stromal compartments. We have shown that the thymic microenvironment can be readily manipulated in vitro through the use of reaggregate thymus organ cultures, which allow the preparation of three-dimensional thymus lobes from defined stromal and lymphoid cells. Although other in vitro systems support some aspects of T-cell development, reaggregate thymus organ culture remains the only in vitro system able to support efficient MHC class I and II-mediated thymocyte selection events, and so can be used as an effective tool to study the cellular and molecular regulation of positive and negative selection in the thymus.
