Single-cell physical phenotyping of mechanically dissociated tissue biopsies for fast diagnostic assessment

Abstract

Rapid and accurate histopathological diagnosis during surgery is critical for clinical decision-making. The prevalent method of intraoperative consultation pathology is time, labour and cost intensive and requires the expertise of trained pathologists. Here, we present an alternative technique for the rapid, label-free analysis of biopsy samples by sequentially assessing the physical phenotype of singularized, suspended cells in high-throughput. This new diagnostic pipeline combines enzyme-free, mechanical dissociation of tissues with real-time deformability cytometry at measurement rates of 100 – 1,000 cells/sec, and machine learning-based analysis. We show that physical phenotype parameters extracted from brightfield images of single cells can be used to distinguish subpopulations of cells in various tissues, without prior knowledge or the need for molecular markers. Further, we demonstrate the potential of our method for inflammatory bowel disease diagnostics. Using unsupervised dimensionality reduction and logistic regression, we accurately differentiate between healthy and tumorous tissue in both mouse and human biopsy samples. The method delivers results within 30 minutes, laying the groundwork for a fast and marker-free diagnostic pipeline to detect pathological changes in solid biopsies.

TissueGrinder, a novel technology for rapid generation of patient-derived single cell suspensions from solid tumors by mechanical tissue dissociation

“…Direct measurement was not performed to minimize the risk of cross-contamination and to demonstrate a consistent and efficient clinical process. Viability results could already be demonstrated in the studies ( 19 , 35 ) with evidence on different tissue samples (spleen, thymus, lymph nodes, liver, kidney, small intestine, colon, lung, stomach, and pancreas). Cells obtained after processing with the TissueGrinder were often more heterogeneous compared to those obtained via the explant method.…”

An enzyme-free, cold-process acoustic method for gentle and effective tissue dissociation

“…While our comparison of murine tissue dissociation measured stark differences in cell surface expression markers across BLU and enzymatic dissociation methods, further work is needed to determine whether the loss of these markers is limited to the cell surface or is indicative of the loss of entire cell populations. Findings of another study, where a comparison of an alternative mechanical dissociation method to the same commercially available enzymatic dissociation kits documented the loss of larger cell populations by enzymatic dissociation, suggest the latter (34). Regardless, our findings demonstrate that important immune cell markers are often compromised or lost by enzymatic dissociation, which may mislead conclusions about immune cell infiltration and localized immune responses.…”

Recent advances in deformation-assisted microfluidic cell sorting technologies

“…Building upon this foundation, there are also several variations of the RT-DC platform. 63,[99][100][101][102] For instance, RT-FDC 99,103 combines the molecular specificity of fluorescent probes with the sensitivity of functional marker cell mechanics. This amalgamation enables researchers to uncover correlations between molecular characteristics and mechanical properties.…”