Comparative framework and adaptation of ACME HS approach to single cell isolation from fresh-frozen endocrine tissues

Abstract

Current scRNA-seq studies of solid tissues mostly rely on enzymatic dissociation of fresh samples or the fallback on nuclei isolation from frozen or partially fixed samples. However, due to the complex tissue organization or cell fragility, it could be challenging to apply these approaches to the sensitive endocrine tissues. That is, dissociating intact cells from such problematic fresh-frozen samples routinely collected by biobanks remains challenging. In this study, we adapted the acetic-methanol dissociation method - ACME High Salt (ACME HS) to effectively isolate intact single cells from fresh-frozen endocrine tumor samples, including adrenal gland neoplasms, thyroid carcinomas, and pituitary neuroendocrine tumors. We compared the ability of enzymatic, ACME HS, and nuclear isolation methods to preserve the integrity of major cell types and gene expression across 41 tissue samples of different origins. We demonstrated that ACME HS simultaneously dissociates and fixes cells, thus preserving morphology and a high RNA integrity number in problematic cell types. This finding renders the ACME HS dissociation method a valuable alternative in scRNA-seq protocols for challenging tissues where obtaining live cell suspension is difficult or impossible.