Isolation, Identification, and Purification of Murine Thymic Epithelial Cells

Summary

Here we describe an efficient method for isolation, identification, and purification of mouse thymic epithelial cells (TECs). The protocol can be utilized for studies of thymus function for normal T cell development, thymus dysfunction, and T cell reconstitution.

Abstract

The thymus is a vital organ for T lymphocyte development. Of thymic stromal cells, thymic epithelial cells (TECs) are particularly crucial at multiple stages of T cell development: T cell commitment, positive selection and negative selection. However, the function of TECs in the thymus remains incompletely understood. In the article, we provide a method to isolate TEC subsets from fresh mouse thymus using a combination of mechanical disruption and enzymatic digestion. The method allows thymic stromal cells and thymocytes to be efficiently released from cell-cell and cell-extracellular matrix connections and to form a single-cell suspension. Using the isolated cells, multiparameter flow cytometry can be applied to identification and characterization of TECs and dendritic cells. Because TECs are a rare cell population in the thymus, we also describe an effective way to enrich and purify TECs by depleting thymocytes, the most abundant cell type in the thymus. Following the enrichment, cell sorting time can be decreased so that loss of cell viability can be minimized during purification of TECs. Purified cells are suitable for various downstream analyses like Real Time-PCR, Western blot and gene expression profiling. The protocol will promote research of TEC function and as well as the development of in vitro T cell reconstitution.

Introduction

Early in T cell development, bone marrow hematopoietic stem cell-derived multipotent progenitors are recruited to the cortex of the thymus, undergo commitment to T lineage and become T cell precursors¹. In the cortex, T cell precursor CD4 and CD8 double negative (DN) thymocytes expand and differentiate into immature CD4 and CD8 double positive (DP) thymocytes, forming a large pool of progenitors with highly variable T cell receptors¹. Only a select MHC-restricted subset of DP cells will become CD4 or CD8 single positive (SP) thymocytes, migrate to the medulla of the thymus, and differentiate into functionally competent mature T cells, an event th....

Protocol

In this study, adult (6 - 8 weeks) female C57Bl/6 mice were used. Mice were purchased from the National Cancer Institute and maintained under specific pathogen free conditions. The University of Minnesota Institutional Animal Care and Use Committee (IACUC) approved all animal experimentation.

1. Preparation of Instruments and Buffers

1. Prepare enzyme solution (RPMI1640 medium with 0.05% [w/v] of Liberase TH and 100 U/ml of DNase I), albumin-rich buffer (1X PBS [Ca²⁺/Mg²⁺-free] with 0.5% Bovine serum albumin and 2 mM Ethylenediamine Tetraacetic acid [EDTA]), FACS buffer (1X PBS [Ca²⁺/Mg²⁺....

Results

Using this protocol, a thymus organ was removed from an adult mouse (Section 2) and a thymic cell suspension was prepared as outlined in Section 3. The obtained cell suspension consisted of thymocytes, hematopoietic-derived stromal cells and non-hematopoietic stromal cells. CD45 is a hematopoietic pan-marker expressed on both thymocytes and hematopoietic stromal cells such as macrophages and dendritic cells. In contrast, TECs are not of hematopoietic origin and do not express CD45¹⁵. Cell staining and flow cyt.......

Discussion

In the protocol, critical steps are the preparation of thymus stromal cells (Section 3) and the enrichment of TECs (Section 4). It is strongly recommended that fresh enzyme solution is prepared each time and the tissue is treated as soon as possible. For pooled thymi, optimizing the volume of enzyme solution is required depending upon the number of thymi. If any tissue residue is left after the processing, adding more enzyme solution and extension of incubation time could increase cell yield. During enrichment of TECs, c.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Anti-mouse EpCAM	eBioscience	XX-5791-YY*	Clone G8.8
Anti-mouse MHC II	eBioscience	XX-5321-YY*	Clone M5/114.15.2
Anti-mouse Ly51	eBioscience	XX-5891-YY*	Clone 6C3
UEA-1	Vector Laboratory	FL-1061
Flow cytometer	BD Biosciences	LSRFortessa
Flow cell sorter	BD Biosciences	FACSAria
FACS tubes	BD Biosciences	352052
Flow cytometry analysis software	TreeStar - Flowjo	FlowJo v7/9
*XX varies by fluorochrome and YY varies by vial size.