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 108 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 × 105 medullary TEC per thymus compared to 5.16 × 104 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.