Reaggregieren Thymus Kulturen

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 diesem Video die Herstellung von 2-dGuo behandelten reaggregieren Thymus Kulturen demonstriert.

Abstract

In der Thymusdrüse, unreifen CD4 +8 + Thymozyten exprimieren vertauschten T-Zell-Rezeptor-α-und b-Ketten-Gene unterziehen positive und negative Selektion Ereignisse auf ihre Fähigkeit, self-peptide/major (MHC)-Moleküle durch Thymus exprimiert erkennen Basis Stromazellen. In vivo Analyse der Rolle des Thymus Stromazellen während intrathymischen Auswahl wird dadurch erschwert, durch die zelluläre Komplexität des Thymus Mikroumgebung in der steady-state adulten Thymus und durch das Fehlen geeigneter Targeting-Strategien, um die Genexpression insbesondere Thymus Stromatumoren Fächer zu manipulieren. Wir haben gezeigt, dass die Thymus-Mikroumgebung leicht in vitro werden durch den Einsatz von reaggregieren Thymus Organkulturen, das die Herstellung von dreidimensionalen Thymus Lappen aus definierten Stroma-und lymphatischen Zellen ermöglichen manipuliert. Auch wenn andere in vitro-Systeme einige Aspekte der T-Zell-Entwicklung zu unterstützen, reaggregieren Thymus Organkultur bleibt die einzige in-vitro-System in der Lage, effiziente MHC-Klasse-Support I und II-vermittelte Thymozyten Auswahl Veranstaltungen und so als ein wirksames Instrument zur Untersuchung verwendet werden kann die zelluläre und molekulare Regulation von positiven und negativen Selektion im Thymus.

Protocol

Für weitere Informationen über die Vorbereitung reggregate Thymus Kulturen besuchen Sie bitte die Springer Protocols .

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.