Abstract 6089: Novel workflow enables enhanced tissue dissociation and gentle clean-up for single cell applications
Authors: Klomsiri Chananat , Grimley Edward , Roussey Jon , Livi Carolina , Wegner Casey , McNaughton Brandon H. , Levers Steve , Vivek Vibhu , Snyder Dan , Summers Doug , Phiengsai Ma 
Published: June 2022

Abstract
With genomic instability being a hallmark of cancer, heterogeneity within solid tumors is prevalent. Intra-tumoral heterogeneity contributes strongly to clinical outcomes. In fact, heterogeneity is often cited as a driver of therapeutic failure, chemo-resistance, and recurrence of disease. Current bulk profiling analysis of tumors is limited in its ability to provide the level of granularity necessary for uncovering aspects of heterogeneity that contribute to poor outcomes. Thus, it is imperative to gain a deeper understanding of tumors at the single-cell level. To properly analyze tumor heterogeneity at the single-cell level it is critical that quality methods for tissue dissociation and cell purification be utilized. While the most frequently used workflows rely on a combination of enzymatic and mechanical dissociation of the tissue followed by RBC lysis to remove contaminating red blood cells, these approaches tend to be harsh on cells leading to activation of cell stress pathways or even the loss of target cells due to their death during processing. The analysis of tumor heterogeneity has long been hampered by issues with low yield and sample preparation-induced alterations in cell physiology. To alleviate these issues, we present a workflow that couples Cellsonics’ SimpleFlow™ with Akadeum’s Buoyancy Activated Cell Sorting (BACS™) Microbubbles for gentle and effective tumor dissociation and clean-up that does not compromise precious samples. Cellsonics’ SimpleFlow™ system features the proprietary Bulk Lateral Ultrasonic™ (BLU™) energy to generate single cells from solid tissue with unparalleled speed, quality, and sample integrity. SimpleFlow™ is engineered to enable any lab to obtain the full spectrum of heterogeneous cell populations while maintaining the native gene expression patterns across all cell types. Single cell samples can be generated from solid tissues in 4-10 minutes under cold temperature without the use of enzymes. Akadeum’s BACS™ Microbubble technology features low-density microbubbles that are functionalized to capture an array of analytes including particular cell types. Akadeum’s BACS™ Microbubbles enable the purification of cell types of interest quickly, easily, and without sacrificing cell health and physiology. In this study, The SimpleFlow™ system was used to dissociate fresh tissues from mice to obtain a heterogeneous suspension of single cells. Following the dissociation, up to 99% of RBCs were removed from samples using Akadeum’s Mouse RBC Depletion. In subsequent studies, cancer cells were spiked into tissue samples prior to processing to assess their recovery and physiology. The novel workflow presented here combing SimpleFlow™ and microbubbles demonstrates the recovery of the full spectrum of cells and did not harm tumor cell recovery or health.
Citation Format: Chananat Klomsiri, Edward Grimley, Jon Roussey, Carolina Livi, Casey Wegner, Brandon H. McNaughton, Steve Levers, Vibhu Vivek, Dan Snyder, Doug Summers, Ma Phiengsai. Novel workflow enables enhanced tissue dissociation and gentle clean-up for single cell applications [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6089.