Preparation and Applications of Organotypic Thymic Slice Cultures

Summary

We describe the preparation of thymic slices that, in combination with flow cytometry, can be used to model positive and negative selection of developing T cells. Thymic slices can also be adapted for the in situ analysis of thymocyte migration, localization, and signaling via immunofluorescence and two-photon microscopy.

Abstract

Thymic selection proceeds in a unique and highly organized thymic microenvironment resulting in the generation of a functional, self-tolerant T cell repertoire. In vitro models to study T lineage commitment and development have provided valuable insights into this process. However, these systems lack the complete three-dimensional thymic milieu necessary for T cell development and, therefore, are incomplete approximations of in vivo thymic selection. Some of the challenges related to modeling T cell development can be overcome by using in situ models that provide an intact thymic microenvironment that fully supports thymic selection of developing T cells. Thymic slice organotypic cultures complement existing in situ techniques. Thymic slices preserve the integrity of the thymic cortical and medullary regions and provide a platform to study development of overlaid thymocytes of a defined developmental stage or of endogenous T cells within a mature thymic microenvironment. Given the ability to generate ~20 slices per mouse, thymic slices present a unique advantage in terms of scalability for high throughput experiments. Further, the relative ease in generating thymic slices and potential to overlay different thymic subsets or other cell populations from diverse genetic backgrounds enhances the versatility of this method. Here we describe a protocol for the preparation of thymic slices, isolation and overlay of thymocytes, and dissociation of thymic slices for flow cytometric analysis. This system can also be adapted to study non-conventional T cell development as well as visualize thymocyte migration, thymocyte-stromal cell interactions, and TCR signals associated with thymic selection by two-photon microscopy.

Introduction

T cells differentiate through a series of developmental intermediates in the thymus during which time they encounter several checkpoints that ensure the generation of a functional, self-tolerant T cell repertoire^1-3. Positive selection promotes the survival of thymocytes with T cell receptors (TCR) capable of recognizing, with low to moderate affinity, peptide presented by major histocompatibility complex molecules (MHC) on cortical thymic epithelial cells (cTEC)^2,3. Negative selection and regulatory T (T_reg) cell development contribute to the establishment of self-tolerance via the elimination or diversion of thymocytes that respond s....

Protocol

Protocols for all animal studies were approved by the Animal Care Committee at the Centre de recherche - Hôpital Maisonneuve-Rosemont.

1. Harvesting Mouse Thymus for Preparation of Thymic Slices and Single Cell Suspensions

1. Euthanize the mouse with CO₂ followed by cervical dislocation.
2. In a laminar flow hood, pin the mouse ventral side up to a dissection board. Spray the mouse with 70% ethanol. Remove any excess alcohol by dabbing with gauze to prevent ethanol from entering the.......

Discussion

Here we describe a protocol for the preparation of thymic slices and representative results of efficient positive and negative selection of overlaid pre-selection MHC class I-restricted TCR transgenic thymocytes by flow cytometry. This system has been used with similar success to support positive selection of MHC class II-restricted CD4⁺ T cells from pre-selection DP thymocytes³², and, in the presence of agonist antigen, negative selection and thymic T_reg development^{11,12,36,38,39,43.......}

Materials

Name	Company	Catalog Number	Comments
Vibratome	Leica Biosystems	VT1000S
NuSieve GTG Agarose	Lonza	50080	Low melting temperature agarose
Embedding Mold (Truncated - T12)	Polyciences	18986	22mm x 22mm square, truncated to 12mm x 12mm
Double Edge Prep Blades	Personna	74-0002
Tissue Adhesive	3M	1469SB
0.4 µm Cell Culture Inserts	BD Falcon	353090	Of several brands tested, these maintained the cells atop the slices the best
Dulbecco's Phosphate-Buffered Saline	ThermoFisher	21600-010
RPMI-1640 with L-glutamine	Wisent	350-000-CL
Fetal Bovine Serum	Wisent	080-110	Heat inactivated
L-Glutamine, 200mM	Wisent	609-065-EL
Penicillin/Streptomycin, 100X	Wisent	450-201-EL
2-Mercaptoethanol	Alfa Aesar	A15890
15 ml Tenbroeck Tissue Grinders	Wheaton	357426
Nylon Mesh Filter	Component Supply	U-CMN-255
Microcentrifuge Tube Sample Pestle	Bel-Art	F19922-0000
40 µm Nylon Cell Strainer	BD Falcon	352340
Forceps Inox Tip	Dumont	RS-5047	Fine tip curved forceps, size .17 X .10mm
Micro Forceps	Dumont	RS-5090