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MRC Center for Immune Regulation, University of Birmingham
White, A., Jenkinson, E., Anderson, G. Reaggregate Thymus Cultures. J. Vis. Exp. (18), e905, doi:10.3791/905 (2008).
胸腺では、未熟なCD4 8 +ランダムに並び替えT細胞受容体α-およびB鎖の遺伝子を発現する胸腺細胞は、胸腺で表さself-peptide/majorの組織適合性複合体（MHC）分子を認識する能力に基づいて正と負の選択イベントを受ける間質細胞。胸腺内選択中の胸腺間質細胞の役割のin vivo解析では定常状態の成人の胸腺における胸腺微小環境の細胞の複雑さによって困難さ、および適切なターゲット戦略の欠如により、特定の胸腺間質コンパートメントにおける遺伝子発現を操作する。我々は胸腺微小環境を容易に定義されている間質およびリンパ系細胞から三次元胸腺葉の準備を可能に再集合させる胸腺器官培養、を使用してin vitroで操作できることを示している。 in vitro系で他はT細胞の開発のいくつかの側面をサポートしていますが、胸腺器官培養は、IとIIを介した胸腺細胞選択イベント、などを勉強するために有効なツールとして使用できる唯一のin vitroの系での効率的なMHCクラスをサポートすることが残って再集合させる胸腺で正と負の選択の細胞および分子調節。
Formal Correction: Erratum: Reaggregate Thymus Cultures
Posted by JoVE Editors on 04/01/2012. 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).
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.