Isolation of Myeloid Dendritic Cells and Epithelial Cells from Human Thymus

Summary

This protocol details a method to isolate antigen presenting cells from human thymus via different steps of enzymatic digestion of the tissue followed by density centrifugation of the single cell suspension and finally magnetic and/or FACS sorting of the cell populations of interest.

Abstract

In this protocol we provide a method to isolate dendritic cells (DC) and epithelial cells (TEC) from the human thymus. DC and TEC are the major antigen presenting cell (APC) types found in a normal thymus and it is well established that they play distinct roles during thymic selection. These cells are localized in distinct microenvironments in the thymus and each APC type makes up only a minor population of cells. To further understand the biology of these cell types, characterization of these cell populations is highly desirable but due to their low frequency, isolation of any of these cell types requires an efficient and reproducible procedure. This protocol details a method to obtain cells suitable for characterization of diverse cellular properties. Thymic tissue is mechanically disrupted and after different steps of enzymatic digestion, the resulting cell suspension is enriched using a Percoll density centrifugation step. For isolation of myeloid DC (CD11c⁺), cells from the low-density fraction (LDF) are immunoselected by magnetic cell sorting. Enrichment of TEC populations (mTEC, cTEC) is achieved by depletion of hematopoietic (CD45^hi) cells from the low-density Percoll cell fraction allowing their subsequent isolation via fluorescence activated cell sorting (FACS) using specific cell markers. The isolated cells can be used for different downstream applications.

Introduction

The thymus is the organ in which T cell development occurs. Its relative and absolute size decreases with age when it becomes successively replaced by fat although thymic activity can still be detected in the old age. Its importance for the immune response was demonstrated in the early 1960s¹.

The T cell repertoire is shaped through the interaction of T cell receptors with peptide-MHC complexes on different kinds of thymic APC, which provide survival or death cues to developing T cells, resulting in a functional and largely self-tolerant T cell repertoire².

Approximately 98% of the cells in ....

Protocol

1. Preparation of Tools, Enzyme Solutions, and Buffers

Perform the following preparative steps prior to beginning the protocol.

1. Tools
   Clean, dry and autoclave the following tools and keep them in sterile packaging until use.
   1. Small sharp scissors with either curved or straight tips for cutting the thymus tissue. Small curved forceps with serrated tips for handling the tissue.
   2. 50 ml Oak Ridge Centrifuge Tubes, PC, for the Percoll-density centrifugation step.
2. Enzyme solutions
   Prepare the Collagenase/DNase I enzyme mix (2 mg Collagenase/ml and 0.1 mg DNase I/ml) as follows....

Results

As starting material in this protocol we use thymus tissue removed from children undergoing corrective cardiovascular surgery (Department of Thoracic and Cardiovascular Surgery, University Clinic Tuebingen) obtained after informed consent and under institutional guidelines. This discarded material can vary greatly in size from 2-30 g or more. The number of mDC and TEC subsets (cTEC and mTEC) that are obtained depends on the size as well as the age of the thymus tissue sample used for isolation.

Discussion

The protocol described here is a modification of the protocol published by Gotter et al⁴. Critical steps in the protocol are the condition and initial preparation of the tissue as well as the Percoll density separation. We strongly recommend to process the tissue as soon as possible after collection. It is important to work fast but thoroughly when cleaning and cutting the tissue. During the thymocyte wash described in step 2.3, it is crucial to find the right balance when applying pressure with the b.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Reagents and Materials
RPMI 1640	PAA	E15-842
Dulbecco's PBS	PAA	H15-002
Fetal Bovine Serum-Gold	PAA	A15-151
Bovine Serum Albumin	PAA	K41-001
Collagenase A	Roche	10 103 586 001
DNase I, grade II bovine pancreatic	Roche	10 104 159 001
Trypsin-EDTA 10x in PBS	PAA	L11-001	stock conc. 20 mg/ml
Alexa Fluor 488 Protein Labelling kit	Molecular Probes	A-10235
anti-human CDR2 (purified)	Bruno Kyewski, DKFZ- Heidelberg, Germany		labeled with Alexa Fluor 488
anti-human CD45 (Pacific Blue)	Biolegend	304022
anti-human EpCAM (APC)	Miltenyi Biotec	130-091-254
anti-human CD11c (PE)	Miltenyi Biotec	130-092-411
anti-PE Microbeads	Miltenyi Biotec	130-048-801
anti-CD45 Microbeads, human	Miltenyi Biotec	130-045-801
LS columns	Miltenyi Biotec	130-042-401
gentleMACS C Tubes	Miltenyi Biotec	130-093-237	for tissue dissociator
Percoll (density 1.130 g/ml)	GE Healthcare, Life Sciences	17-0891-01
Sterile distilled Water (DNAse/ RNAse free)	GIBCO	10977-035
Gamunex 10%	Tajecris-Biotherapeutics	G120052	1:10 pre-dilution, use 20 μl/1 x 106cells
0.22 μm filter	Millex GS	SLGS033SS	Syringe driven
Stericup filter unit	Millipore	SCGPU05RE	Pump driven
50 ml PC oak ridge centrifuge tubes	Nalgene	3118-0050	50 ml
50 ml PP conical tubes	Becton Dickinson	352070
12 mm x 75 mm 5 ml test tubes	Becton Dickinson	352058	FACS stainings
Cell strainer 70 μm	Becton Dickinson	352350
INSTRUMENTS
Flow Cytometer-Sorter (BD FACSAriaTMIIu)	Becton Dickinson
Sorvall Evolution R6 (rotor)	Kendro
Rotator REAX 2	Heidolph
gentleMACS Dissociator	Miltenyi Biotec	130-093 235	tissue dissociator