Reaggregate thymus cultures.
ABSTRACT 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].
Full-textDOI: · Available from: Graham Anderson, Sep 02, 2014
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- "Attempts to engineer an artificial thymus have mostly used traditional tissue engineering approaches involving chemical scaffolds and cells liberated from isolated tissues, although newer methods involving decellularised organs show some potential. More simplistic methods to 'engineer' a thymus are foetal thymus organ culture (FTOC) and reaggregate thymus organ culture (RTOC) (Figure 2) (Anderson and Jenkinson 2007; White et al. 2008). These are both methods whereby the foetal thymus is ablated of T cell precursors prior to addition of HSC derived cells. "
ABSTRACT: Tissue engineering is making great strides to repair disease and damage in a wide range of tissues, many of which are high profile and well documented. However, the thymus and secondary lymphoid organs are tissues which have not received significant attention from the research community but are nonetheless important targets for tissue engineering based therapies. These organs are fundamental in developing and maintaining the mammalian immune system and create environments for antigen screening and pathogen removal. This review discusses the function of these organs with reference to tissue development, tissue resident progenitor cells and disease. Subsequently strategies utilised for tissue engineering and regeneration are discussed in conjunction with methods to replicate their function and alternative methods to generate progenitor cells. Introduction Tissue engineering is a multidisciplinary research field which aims to reproduce, repair or augment tissue function of whole or partial organs in individuals suffering disease or trauma. The field has roots in clinical medicine and employs the principles of engineering and materials chemistry with cell biology. This involves the selective use of cells, scaffold biomaterials, growth factors and mechanical stimulation in various combinations to recapitulate an organ in vitro or in vivo. It was first mentioned as a distinct area of research in the 1980's, before coming to prominence through Langer and Vacanti's seminal 1993 article Tissue Engineering (Skalak and Fox 1988; Langer and Vacanti 1993). Since this point thousands of research articles have been published on engineering or repairing practically every organ of the human body, with particular focus on musculoskeletal tissues (2201 publications in 2012), the heart (422) and skin (314). Two groups however, have received comparatively little attention; these are the primary and secondary lymphoid organs (SLO) (less than 10 specific articles in 2012). The primary lymphoid organs are composed of the bone marrow and the thymus, with the thymus being the major site of T cell generation, whilst the SLO comprise lymph nodes and lymphoid follicles in organs such as the spleen, tonsils and adenoids. As a group these organs generate and maintain the mammalian immune system and are fundamental to survival. Despite the compa-rative lack of effort advances have been made in attempting to regenerate these organs or recapitulate their functions and these will be discussed.05/2013; 1(5):12-28. DOI:10.14304/SURYA.JPR.V1N5.3
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ABSTRACT: Thymic epithelial cells (TECs) provide a highly specialized microenvironment for the generation of a functional and self-tolerant T cell repertoire. Much of our current view of TEC biology is derived from gain- or loss-of-function approaches, which have significantly contributed to our understanding of gene function in TEC development and T cell repertoire selection. Here, we will review transgenic and viral strategies that have been used to manipulate gene expression in TECs, highlight some of the shortcomings of particular currently available tools and provide a brief outline of our own attempts to more rapidly and/or more specifically assess gene function in TECs.European journal of cell biology 03/2011; 91(1):24-30. DOI:10.1016/j.ejcb.2011.01.005 · 3.70 Impact Factor
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ABSTRACT: The vitamin A metabolite and transcriptional modulator retinoic acid (RA) is recognized as an important regulator of epithelial cell homeostasis in several tissues. Despite the known importance of the epithelial compartment of the thymus in T cell development and selection, the potential role of RA in the regulation of thymic cortical and medullary epithelial cell homeostasis has yet to be addressed. In this study, using fetal thymus organ cultures, we demonstrate that endogenous RA signaling promotes thymic epithelial cell (TEC) cell-cycle exit and restricts TEC cellularity preferentially in the cortical TEC compartment. Combined gene expression, biochemical, and functional analyses identified mesenchymal cells as the major source of RA in the embryonic thymus. In reaggregate culture experiments, thymic mesenchyme was required for RA-dependent regulation of TEC expansion, highlighting the importance of mesenchyme-derived RA in modulating TEC turnover. The RA-generating potential of mesenchymal cells was selectively maintained within a discrete Ly51(int)gp38(+) subset of Ly51(+) mesenchyme in the adult thymus, suggesting a continual role for mesenchymal cell-derived RA in postnatal TEC homeostasis. These findings identify RA signaling as a novel mechanism by which thymic mesenchyme influences TEC development.The Journal of Immunology 04/2012; 188(10):4801-9. DOI:10.4049/jimmunol.1200358 · 5.36 Impact Factor