A novel method to efficiently isolate medullary thymic epithelial cells from murine thymi based on UEA-1 MicroBeads.

OBJECTIVE

The central mechanism for establishing a self-tolerant and functional T cell repertoire includes the promiscuous expression of otherwise tissue-restricted proteins by medullary thymic epithelial cells (TEC). We here demonstrate a novel and highly efficient method for isolating this rare key cell type.

METHODS

We combined the enrichment of medullary TEC via UEA-1 MicroBeads with the subsequent depletion of residual CD45 hematopoietic cells via specific size exclusion and compared our results to the standard Percoll enrichment method and isolation procedure via flow cytometric cell sorting.

RESULTS

The addition of 2 μl UEA-1 MicroBeads per 10 thymus cells turned out best for optimal enrichment (an average of 22% purity compared to 1.2% for Percoll) and yield (an average of 1.73 × 10 medullary TEC per thymus compared to 5.16 × 10 for Percoll). After depletion of residual CD45 cells, our method not only reached a purity of 75.5% but also turned out less stressful for the cells as compared to flow cytometric cell sorting.

CONCLUSION

We here provide a fast and versatile procedure for enriching medullary TEC that yields higher purity and recovery rates than the standard Percoll enrichment method Our enrichment procedure in combination with CD45 depletion via specific size exclusion is comparable to the current gold standard flow cytometric cell sorting method.

SIGNIFICANCE STATEMENT

We developed a fast and versatile procedure to isolate a high number medullary TEC to investigate the biochemical processes of medullary TEC in more depths.