Thymic epithelial cells (TEC) are a critical component of the three-dimensional (3D) thymic stromal network mediating T-cell development and self-tolerance. Models supporting the culture of functionally competent adult TEC in vitro are currently lacking, hampering thymus engineering strategies and a deeper understanding of the development of a competent immune system. Therefore, we aimed to establish a 3D model based on decellularized thymus for ex vivo culture of functional TEC. A perfusion bioreactor-based protocol for decellularization of murine thymus was developed. Efficient DNA removal was verified by histology and PicoGreen assay. The main components of the thymus native extracellular matrix were preserved, as shown by immunofluorescence for collagen IV, laminin and fibronectin. The decellularized tissue was mechanically disrupted, lyophilized and cross-linked to produce 3D porous scaffolds (thymus scaffold, TS). Microstructure was characterized via scanning electron microscopy. TS supported adult TEC culture in vitro, with preserved TEC phenotype at 2 weeks (immunofluorescence staining, mostly K14+). Fetal TEC cultured with T cell precursors in TS supported thymocyte differentiation in vitro. TS were then seeded with TEC isolated from adult mice and grafted under the kidney capsule of athymic nude mice. Host derived CD4+/CD8+ double positive thymocytes were detected in the graft, and host-derived CD4+ or CD8+ T cells were found in lymph nodes after 7 weeks, demonstrating graft-derived thymopoiesis in vivo. We have developed a 3D model that supports culture and functionality of adult TEC, paving the way to a better understanding of TEC/thymocytes cross-talk and to unprecedented approaches for the treatment of thymic-related pathologies.

Decellularized Thymus for In Vitro Culture of Functional Adult Thymic Epithelial Cells / Asnaghi, Ma; Barthlott, T; Strusi, V; Toni, R; Hollander, G; Martin, I; Wendt, D. - In: TISSUE ENGINEERING, PART A. - ISSN 1937-3341. - 21(2015), pp. 275-275.

Decellularized Thymus for In Vitro Culture of Functional Adult Thymic Epithelial Cells

Toni, R;Martin, I
Membro del Collaboration Group
;
2015

Abstract

Thymic epithelial cells (TEC) are a critical component of the three-dimensional (3D) thymic stromal network mediating T-cell development and self-tolerance. Models supporting the culture of functionally competent adult TEC in vitro are currently lacking, hampering thymus engineering strategies and a deeper understanding of the development of a competent immune system. Therefore, we aimed to establish a 3D model based on decellularized thymus for ex vivo culture of functional TEC. A perfusion bioreactor-based protocol for decellularization of murine thymus was developed. Efficient DNA removal was verified by histology and PicoGreen assay. The main components of the thymus native extracellular matrix were preserved, as shown by immunofluorescence for collagen IV, laminin and fibronectin. The decellularized tissue was mechanically disrupted, lyophilized and cross-linked to produce 3D porous scaffolds (thymus scaffold, TS). Microstructure was characterized via scanning electron microscopy. TS supported adult TEC culture in vitro, with preserved TEC phenotype at 2 weeks (immunofluorescence staining, mostly K14+). Fetal TEC cultured with T cell precursors in TS supported thymocyte differentiation in vitro. TS were then seeded with TEC isolated from adult mice and grafted under the kidney capsule of athymic nude mice. Host derived CD4+/CD8+ double positive thymocytes were detected in the graft, and host-derived CD4+ or CD8+ T cells were found in lymph nodes after 7 weeks, demonstrating graft-derived thymopoiesis in vivo. We have developed a 3D model that supports culture and functionality of adult TEC, paving the way to a better understanding of TEC/thymocytes cross-talk and to unprecedented approaches for the treatment of thymic-related pathologies.
Decellularized Thymus for In Vitro Culture of Functional Adult Thymic Epithelial Cells / Asnaghi, Ma; Barthlott, T; Strusi, V; Toni, R; Hollander, G; Martin, I; Wendt, D. - In: TISSUE ENGINEERING, PART A. - ISSN 1937-3341. - 21(2015), pp. 275-275.
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Utilizza questo identificativo per citare o creare un link a questo documento: http://hdl.handle.net/11381/2837620
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