Comparison of processing approaches for single-cell analysis of esophageal biopsy samples.

Background: Eosinophilic esophagitis is an eosinophil-rich type 2 inflammatory disease with increasing global prevalence. Multicenter randomized trials are critical, but use of certain cutting-edge techniques, such as single-cell sequencing, can be challenging owing to a requisite need for immediate tissue processing. Methods: We performed a pilot single-cell RNA sequencing (scRNA-seq) study using single esophageal biopsy samples in 3 processing protocols. Biopsy samples were processed as (1) fresh, (2) immediately frozen, or (3) frozen after dispersion to single cells. scRNA-seq was performed using 10× Genomics to understand cell type preservation despite freezing. Results: Biopsy samples from 4 patients were processed with 1 or more of each of the fresh and frozen protocols. Comparison of the scRNA-seq profiles of frozen samples with those obtained using the criterion standard of fresh biopsy samples demonstrated that epithelial and mast cell transcriptomes were preserved in frozen dispersed cells and frozen tissues. Lymphocytes, myeloid cells, endothelial cells, and fibroblasts were present in all of the fresh samples. Fibroblast, myeloid, and endothelial cells were lost in immediately frozen tissue but relatively preserved in tissue dispersed to single cells and then frozen. All 9 epithelial cell clusters present in the fresh samples were preserved in dispersed frozen cells but variably retrieved from directly frozen tissue. Conclusion: Single esophageal biopsy specimens can be used successfully in scRNA-Seq when samples are dispersed and frozen as single cells. Myeloid cell, endothelial cell, and fibroblast transcriptomes, as well as nondifferentiated epithelial cell transcriptomes, are not preserved when tissue samples are frozen without initial dispersion to single cells.