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 (CD45hi) 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 the human thymus are developing T cells referred to as thymocytes. The remaining 2% consist of a number of different cell types, including a variety of TEC (cortical, medullary, subcapsular), myeloid and plasmacytoid DC (mDC, pDC), macrophages, B cells, mature re-circulating T cells, granulocytes, fibroblasts, endothelial cells and very rare epithelial cells with an expression phenotype resembling that of cells from other tissues such as muscle, neurons and respiratory epithelium (Figure 1). Of these, TEC and DC are the major APC types found in a normal thymus. In recent years, purification of these APC types for culture and molecular profiling has gained more and more interest. Due to their low frequency, isolation of any of these cell types for detailed analysis requires an efficient, reproducible and cost-effective procedure. The method presented here is a modification from previously published studies3,4.
As with any other tissue, cell extraction from the thymus can be achieved by enzymatically disaggregating the cell-cell and cell-matrix interaction networks, in order to obtain a suspension of single cells. There are certain parameters like good dissociation efficiency, cell yield, cell viability and retention of cell surface markers that are crucial and need to be optimized for the successful isolation of these rare cell populations.
In this protocol, isolation of DC and TEC subsets is performed by making a single-cell suspension of the tissue by mechanical disruption and enzymatic digestion. We use Collagenase A from Clostridium histolyticum, which has a balanced ratio of different enzyme activities, to break down the native collagen that holds the tissue together. DNase I is included in the enzyme solution to reduce cell aggregation due to free DNA from dead cells (thymocytes are very sensitive).We also provide an alternative approach to the typical enzymatic tissue digestion involving mechanical and enzymatic tissue treatment assisted by a tissue dissociator. The single cell suspension is then subjected to a single Percoll density centrifugation for enrichment of low density fraction (LDF) of cells. From this fraction of cells, DC can be isolated by staining for DC-surface markers (i.e. CD11c+) and using magnetic separation or fluorescence-activated cell sorting (FACS). Unlike the lymphoid cells comprising the vast majority of cells in the thymus, TEC do not express CD45 at high levels, but are positive for the epithelial cell adhesion molecule EpCAM. cTEC can be distinguished from medullary TEC by the expression of a yet undefined antigen recognized by the CDR-2 (cortical dendritic reticulocyte-2) antibody4,5 and somewhat lower EpCAM expression. The differential co-expression of EpCAM and CDR2 allows the efficient isolation of these TEC subsets via high-speed cell sorting6.
The protocol presented here is optimized for human thymic tissue. The duration of the procedure depends on the amount of tissue and the ability of the experimenter as well as the speed of the cell sorter, if FACS sorting is used. Normally, the protocol for the isolation of DC can be completed within 5-6 hr and for the isolation of TEC in 8-10 hr. The isolation of DC and TEC subsets from thymic tissue is time sensitive. The faster the isolation procedure, the better the condition of the cells. Finally, the isolated cells can be used for further investigations like comparative studies of mRNA and protein expression, PCR experiments, protein isolation, molecular profiling (i.e. transcriptomics, micro RNA analysis) as well as cell culture6.
Ethics Statement
In order to be able to work with human thymus tissue the researcher needs to obtain approval from the local ethics committee or responsible authorities as well as an informed written consent of the donor (or usually his or her parents, since tissue is usually obtained from underage children). Furthermore, all human tissues should be handled as being potentially infectious and appropriate measures should be taken, such as working with gloves, etc.
Protocol
Representative Results
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…
Disclosures
The authors have nothing to disclose.
Acknowledgements
We are grateful to the surgeons of the Department of Thoracic and Cardiovascular Surgery, University Clinic Tuebingen for providing us with the thymus samples and Bruno Kyewski (DKFZ, Heidelberg, Germany) for providing the CDR2 antibody. We would also like to thank Hans-Jörg Bühring and Sabrina Grimm from the sorting facility (University of Tuebingen). This work was supported by the SFB 685 and the Hertie Foundation.
Materials
| 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 |
References
- Miller, J. F. A. P. The discovery of thymus function and of thymus-derived lymphocytes. Immunological Reviews. 185, 7-14 (2002).
- Klein, L., Hinterberger, M., Wirnsberger, G., Kyewski, B. Antigen presentation in the thymus for positive selection and central tolerance induction. Nat Rev Immunol. 9, 833-844 (2009).
- Vandenabeele, S., Hochrein, H., Mavaddat, N., Winkel, K., Shortman, K. Human thymus contains 2 distinct dendritic cell populations. Blood. 97, 1733-1741 (2001).
- Gotter, J., Brors, B., Hergenhahn, M., Kyewski, B. Medullary Epithelial Cells of the Human Thymus Express a Highly Diverse Selection of Tissue-specific Genes Colocalized in Chromosomal Clusters. The Journal of Experimental Medicine. 199, 155-166 (2004).
- Rouse, R. V., Bolin, L. M., Bender, J. R., Kyewski, B. A. Monoclonal antibodies reactive with subsets of mouse and human thymic epithelial cells. Journal of Histochemistry & Cytochemistry. 36, 1511-1517 (1988).
- Stoeckle, C., et al. Cathepsin S dominates autoantigen processing in human thymic dendritic cells. Journal of Autoimmunity. 38, 332-343 (2012).
- Woods Ignatoski, K. M., Bingham, E. L., Frome, L. K., Doherty, G. M. Directed trans-differentiation of thymus cells into parathyroid-like cells without genetic manipulation. Tissue Eng Part C Methods. 17, 1051-1059 (2011).
- Williams, K. M., et al. Single Cell Analysis of Complex Thymus Stromal Cell Populations: Rapid Thymic Epithelia Preparation Characterizes Radiation Injury. Clinical and Translational Science. 2, 279-285 (2009).
- Bendriss-Vermare, N., et al. Human thymus contains IFN-α-producing CD11c-, myeloid CD11c+, and mature interdigitating dendritic cells. The Journal of Clinical Investigation. 107, 835-844 (2001).
- Schmitt, N., et al. Ex vivo characterization of human thymic dendritic cell subsets. Immunobiology. 212, 167-177 (2007).
- Dzionek, A., et al. BDCA-4: Three Markers for Distinct Subsets of Dendritic Cells in Human Peripheral Blood. The Journal of Immunology. 165, 6037-6046 (2000).
- Wu, L., Shortman, K. Heterogeneity of thymic dendritic cells. Seminars in Immunology. 17, 304-312 (2005).
- Seach, N., Wong, K., Hammett, M., Boyd, R. L., Chidgey, A. P. Purified enzymes improve isolation and characterization of the adult thymic epithelium. Journal of Immunological Methods. 385, 23-34 (2012).
- Adamopoulou, E., Tenzer, S., Hillen, N., Klug, P., Rota, I. A., Tietz, S., Gebhardt, M., Stevanovic, S., Schild, H., et al. Exploring the MHC-peptide matrix of central tolerance in the human thymus. Nature Communications. , (2013).
