CytoR1™ Platform Enriches Samples’ Viability for Single-Cell Sequencing and Downstream Assays

DE Thomas, KE Degen, R Barua, D Keck, AR Hyler. CytoR1 Platform enriches samples’ viability for single-cell sequencing and downstream assays. American Society for Cell Biology Annual Meeting, Washington D.C. December 2022.

Maintaining stable sample heterogeneity with sample co-cultures during dead cell removal and sample enrichment assays are an important process for DNA and RNA sequencing, protein analyses, and phenotypic studies. These single-cell level studies require an input sample with a high rate of viability. If too many dead cells are present in the input, degraded proteins and ambient DNA or RNA can increase background noise that may lead to missing identification of crucial targets. Since the cost for single-cell sequencing experiments remains high, it is critical to ensure the input sample is optimized for high viability to ensure cost-effective and reproducible data. Current bead assays or other sample viability enrichment techniques used in preparation for single-cell analyses typically result in a significant sample loss and bias of sample heterogeneity. These beads or kits can be biased and change the cell population, subject cells to unwanted stresses, and diminish sample integrity with the time needed for preparations. Here, we investigate sample cell viability enrichment on the Cyto R1 Platform, a label-free, cell enrichment, sorting, and recovery platform. At the core, the Cyto R1 uses Cyto Chips, microfluidic technologies utilizing contactless dielectrophoresis (cDEP), to phenotypically enrich and sort various cells based upon unique physical structures and subcellular features. Thus, the Cyto R1 ensures native cell recovery without any unwanted cell tagging to maximize sample integrity. Initial experiments characterized the change in concentration for several co-cultures undergoing batch processing at a voltage of 600 Vpp and frequency of 300 kHz on the Cyto R1 Platform. Co-cultures included combinations of EL4, stem cell population, slow-developing ovarian cancer, fibroblasts, THP-1 monocytes, highly aggressive ovarian cancer, and K562 cells. For all cell types listed the change in concentration of a single species in the co-culture was less than 5%. Notably the change in concentration was less than 2% for THP-1 cells, FFLs, and K562 natural killer cells in their respective co-culture. In parallel studies comparing the EL4 cells batched on the Cyto R1 platform to the current industry standard, magnetic bead assays, the magnetic bead assays altered the heterogeneity of the sample by an average of 20% more when compared to the Cyto R1 Platform. Advancements are being made to characterize the change in concentration of heterogenous cultures for different batch voltages and frequencies to ensure that the sample heterogeneity and viability are maintained.

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