Reaggregate Culture Thymus

Published: August 28, 2008 doi: 10.3791/905

Andrea White¹, Eric Jenkinson¹, Graham Anderson¹

¹MRC Center for Immune Regulation, University of Birmingham

ERRATUM NOTICE

Important: There has been an erratum issued for this article. Read more …

Summary

In questo video la preparazione di 2-dGuo trattati reaggregate culture timo è dimostrata.

Abstract

Nel timo, immaturi CD4 +8 + timociti che esprimono in modo casuale riarrangiati cellule T recettori α-e b-catena di geni sottoposti a selezione di eventi positivi e negativi in base alla loro capacità di riconoscere self-peptide/major complessi di istocompatibilità (MHC) molecole espressa dal timo cellule stromali. L'analisi in vivo del ruolo di cellule stromali del timo durante la selezione intratimica è resa difficile dalla complessità cellulare del microambiente timico in steady-state timo adulto, e dalla mancanza di adeguate strategie di targeting per manipolare l'espressione genica, in particolare, comparti timica stromale. Abbiamo dimostrato che il microambiente timica può essere facilmente manipolate in vitro tramite l'uso di reaggregate culture organo del timo, che permettono la preparazione di lobi timo tridimensionale definito da cellule stromali e linfoidi. Anche se gli altri sistemi in vitro supportano alcuni aspetti di cellule T sviluppo, reaggregate coltura d'organo thymus rimane l'unico sistema in vitro in grado di supportare efficienti MHC di classe I e II-mediata eventi di selezione timocita, e quindi può essere utilizzato come uno strumento efficace per lo studio la regolazione cellulare e molecolare di selezione positiva e negativa nel timo.

Protocol

Per ulteriori informazioni sulla preparazione reggregate culture timo si prega di visitare protocolli Springer .

Erratum

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).

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.