3D Organotypic Co-culture Model Supporting Medullary Thymic Epithelial Cell Proliferation, Differentiation and Promiscuous Gene Expression

Abstract

Intra-thymic T cell development requires an intricate three-dimensional meshwork composed of various stromal cells, i.e., non-T cells. Thymocytes traverse this scaffold in a highly coordinated temporal and spatial order while sequentially passing obligatory check points, i.e., T cell lineage commitment, followed by T cell receptor repertoire generation and selection prior to their export into the periphery. The two major resident cell types forming this scaffold are cortical (cTECs) and medullary thymic epithelial cells (mTECs). A key feature of mTECs is the socalled promiscuous expression of numerous tissue-restricted antigens. These tissue-restricted antigens are presented to immature thymocytes directly or indirectly by mTECs or thymic dendritic cells, respectively resulting in self-tolerance.Suitable in vitro models emulating the developmental pathways and functions of cTECs and mTECs are currently lacking. This lack of adequate experimental models has for instance hampered the analysis of promiscuous gene expression, which is still poorly understood at the cellular and molecular level. We adapted a 3D organotypic co-culture model to culture ex vivo isolated mTECs. This model was originally devised to cultivate keratinocytes in such a way as to generate a skin equivalent in vitro. The 3D model preserved key functional features of mTEC biology: (i) proliferation and terminal differentiation of CD80 lo , Aire-negative into CD80 hi , Aire-positive mTECs, (ii) responsiveness to RANKL, and (iii) sustained expression of FoxN1, Aire and tissue-restricted genes in CD80 hi mTECs. Video LinkThe video component of this article can be found at

The absence of the autoimmune regulator gene (AIRE) impairs the three-dimensional structure of medullary thymic epithelial cell spheroids

“…Several strategies have been developed to study the functional thymic properties, including ex vivo reaggregation thymic organ culture (RTOC) [19, 20], 2D mTEC-thymocyte adhesion [17, 18], transwell thymocyte migration [29], 3D fetal thymus organ culture (FTOC) [30], and 3D organotypic culture [23]. Considering that the 3D intrathymic environment is necessary for the development of a functional thymus scaffolding, development of thymic epithelial cells, chemoattraction of pro-T-cells, commitment to the T lineage, and generation of a repertoire of T-cell receptors (TCR) and natural T regulatory cells [31-33], an adequate thymus structure is mandatory to functionality study mTEC-mTEC interactions.…”

“…Two experimental strategies enable the study of the in vitro formation of the thymus’s 3D structure that reproduces its microenvironment with the extracellular matrix and thymic epithelial cells. One of these strategies is the thymus re-aggregation [19, 20], and the other is the 3D organotypic culture [23]. In the re-aggregation model, the thymus tissue is devoid of cells (decellularized).…”

Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma

“…To create the dermal equivalent (DE), human fibroblasts were cultured on a viscose fiber fabric, that was punched in circles of 11mm in diameter and autoclaved in advance. These constructs grew in 12-well plates with hanging inserts in DMEM medium (4g glucose/l//Ham’s F12: 1:1 (500 mL + 500 mL), 10% FCS, 2% Pen/Strep, hydrocortisone (400 µg/L), ascorbic acid (50 µg/mL), aprotinin (250 E/mL)) [ 24 , 25 , 26 ] for up to 14 days prior to tumor cultivation, allowing fibroblasts to produce extracellular matrix and hence form solid tissue matrices (DEs).…”

Thymus Degeneration and Regeneration

“…Additionally, thymus transplantation and in vitro bioengineering can either be utilized to boost the recovery of thymic function or treatment of patients with congenital thymic atrophy ( 184 ). The most commonly used method is the transplantation of artificial thymic stromal cells, mainly thymic epithelial progenitor cells (TEPC), containing intercellular interaction components to support T cell development ( 185 ). Studies of animal models have demonstrated that, via an in vitro reprogramming technique, TEPC can induce human embryonic stem cells to form TEC and then transplant them into mice under the regulation of FOXN1, IL-7 ( 130 , 156 ), BMP4 ( 78 ), FGF and EGF to form thymus structure ( 155 , 185 ).…”

“…The most commonly used method is the transplantation of artificial thymic stromal cells, mainly thymic epithelial progenitor cells (TEPC), containing intercellular interaction components to support T cell development ( 185 ). Studies of animal models have demonstrated that, via an in vitro reprogramming technique, TEPC can induce human embryonic stem cells to form TEC and then transplant them into mice under the regulation of FOXN1, IL-7 ( 130 , 156 ), BMP4 ( 78 ), FGF and EGF to form thymus structure ( 155 , 185 ). Another method is to remove all the thymus cells and leave only the matrix components, which can be recombined with artificial thymic stromal cells and T lymphoid progenitor cells to form functional thymus ( 186 ).…”