Abstract 2109: Comparison of four non-enzymatic, mechanical dissociation methods for formalin-fixed tissue in flow cytometric analysis and single-cell workflows
Authors: Hannah L. Veloz, Samantha M. Hill, Gloria Asantewaa, Lisa L. Gallegos, Nelson R. Alexander
Journal: Cancer Research (2025)

Abstract Introduction: Formalin-fixed tissue dissociation is a common method to isolate single cells from tissues that can be used for various downstream applications such as flow cytometry or other single-cell workflows. Common dissociation methods often use enzymes to break down tissue; however, this approach can inconsistently damage cell membranes, limiting the expression of biomarkers detectable by flow cytometric analysis. Non-enzymatic tissue dissociation preserves cell epitopes for optimal antibody recognition. In this study, we provide a comparison of four different mechanical dissociation methods and determine how each method affects cell integrity. Methods: Surgically resected, formalin-fixed breast and colon tumors were processed using the representative sampling method and the non-tumor tissue was further homogenized into single cells using four different mechanical dissociation methods. The mechanical methods used in this comparison were the IKA tube mill, Miltenyi C tube, hand-held cordless homogenizer, and a pestle. Prior to mechanical dissociation, the tissue was incubated in a cell conditioning buffer. Single cell suspensions were quantified using the Countess II, and equal cell inputs were stained in suspension for Cytokeratin 8/18, CD45, CD68, CD163. DNA content was assessed via DAPI staining. Samples were analyzed on BD FACSMelody and data analysis was performed using FCS Express 7 software. Results: The four non-enzymatic, mechanical tissue dissociation methods were successful at generating single cell suspensions with no significant difference in the total cell counts. Flow cytometric analysis showed no significant difference in singlet generation visualized by DAPI. There was no significant difference in the percentage of CD68 and CD163 positive populations seen via flow. A statistically non-significant difference was observed in the percentage of CD45 positive and Cytokeratin 8/18 positive populations, indicating that certain methods might be better at liberating immune vs tumor cells. Conclusion: We demonstrate that four non-enzymatic, mechanical tissue dissociation methods are capable of liberating single cells from formalin-fixed tissues. Preservation of cell surface biomarkers is a significant improvement from isolation of nuclei that enables single-cell suspensions to be analyzed using fluorescently labeled antibodies and the enrichment of cell populations of interest via fluorescence activated cell sorting (FACS). Single cell generation and analysis of intact single cells from formalin fixed tissue is novel, and increases downstream applications that can be useful in the research and translational space, and may have clinical applications in the future. Citation Format: Hannah L. Veloz, Samantha M. Hill, Gloria Asantewaa, Lisa L. Gallegos, Nelson R. Alexander. Comparison of four non-enzymatic, mechanical dissociation methods for formalin-fixed tissue in flow cytometric analysis and single-cell workflows. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2025; Part 1 (Regular Abstracts); 2025 Apr 25-30; Chicago, IL. Philadelphia (PA): AACR; Cancer Res 2025;85(8_Suppl_1):Abstract nr 2109.