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Immunology and Infection

Preparation and Applications of Organotypic Thymic Slice Cultures

Published: August 6th, 2016

DOI:

10.3791/54355

1Centre de Recherche, Hôpital Maisonneuve-Rosemont, 2Department of Microbiology, Infectiology and Immunology, Université de Montréal, 3Department of Medicine, Université de Montréal
* These authors contributed equally

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.

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.

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 repertoire1-3. 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 (Treg) cell development contribute to the establishment of self-tolerance via the elimination or diversion of thymocytes that respond s....

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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.......

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Thymic slices support analysis of different aspects of T cell development such as positive and negative selection. For successful experiments, the quality of the thymic slice is paramount. Thus, thymic slices should be examined to ensure the integrity of the thymic tissue and that the agarose surrounding the thymic slice is intact (Figure 1A). Surface tension can be compromised when the agarose is damaged causing a significant decrease in the number of th.......

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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 Treg development11,12,36,38,39,43.......

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We would like to thank Marilaine Fournier for her comments on the manuscript and Josée Tessier for technical assistance. C57BL/6-Tg (OT-I)-RAG1<tmMom> #4175 were obtained through the NIAID Exchange Program, NIH. Support for this research is provided by a grant from the SickKids Foundation and CIHR-IHDCYN (NI15-002), an operating grant from the CIHR-III (MOP-142254), and start-up funds from the FRQS (Établissement de jeunes chercheurs) and Hôpital Maisonneuve-Rosemont Foundation to HJM. HJM is a junior 1 scholar of the FRQS, a CIHR New Investigator (MSH-141967), and a Cole Foundation Early Career Transition award recipient.

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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 

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