Delivering high quality single-cell analysis
Single-cell analysis technologies have facilitated significant developments and discoveries in translational biology. Continued growth and transformation of the tools surrounding the single-cell workflow are required to meet the high bar for quality data produced and quantity of useable data per experiment.
Scientists often discover these issues too late in the experimental pipeline, which contributes to increasing frustration and unanticipated costs in irreplaceable lost samples, time, resources and funds.
Enable samples to tell their native truth by maximizing the data obtained from each and every cell analyzed.
Levitation Technology
LevitasBio® has developed an innovative Levitation Technology that runs a touch-free, label-free, three-step enrichment process on up to 96 samples at once with the LeviCell 96, or 12 with the LeviCell 12, with no centrifuge or beads. This can be applied seamlessly to a wide range of research areas and workflows.
Step 1: Input
Mix each sample with Levitation Agent, a paramagnetic solution, and pipette it into the wells of the SALT™ levitation array: up to 96 samples on the LeviCell 96 or 12 on the LeviCell 12.
Step 2: Enrichment
Place the array in a standard SBS deck position. In every well, viable cells levitate into a tight band above dead cells and debris during a 40-minute levitation, with no labels, beads or centrifuge.
Step 3: Collection
The liquid handler collects the enriched fraction from every well into an output plate, ready for counting and downstream assays, leaving dead cells, debris and supernatant behind.
*Illustrations show the LeviCell 96 System. The LeviCell 12 runs the same three-step workflow and chemistry for up to 12 samples per run.
Viable cells levitate much higher than dead cells and debris, allowing for clear separation and collection by the LeviCell systems. Start with high-quality cells every time for the best and most accurate results. This gentle yet effective process makes it possible.
- Maximize cell viability and enrichment
- Preserve cell population representation
- Minimize the risk of contamination and damage
- Maximize data for every sample analyzed
Remove the noise and maximize data that matters
The innovative label-free separation technology of the LeviCell systems facilitate complete debris and dead cell removal without affecting the original population representation of gene expression (please see our webinar summary).
When it comes to single-cell study, purity and viability of cells harvested from the sample preparation stage is a determining factor for generating high quality data in downstream assays such as NGS (Next Generation Sequencing).
Case study: 10x Genomics single cell sequencing
The 10x Genomics single-cell sequencing platforms have helped to revolutionize our understanding of biology and how the measurement of individual cells’ gene expression can uncover the previously hidden messages commonly unseen in bulk mRNA experiments.
Unfortunately while the single cell sequencing workflow has proven to be fast, simple and reliable, obtaining the most complete and accurate single cell data depends on up-front sample preparation steps. Traditional methods currently in use tend to damage cells, have low capture efficiency, and often completely fail to process sensitive and difficult to work with samples such as dissociated tissue samples. The most common sample enrichment methods, flow cytometry and bead-based enrichment methods, both often lead to significant cell stress and gene expression changes due to the high pressures used (Xiong et al., 2002; Romero-santacreu et al., 2009; Van Den Brink et al., 2017) or cell signaling effects due to the cell surface binding required by these methods (Kornbluth and Hoover, 1989; Christaki et al., 2011). Due to these shortcomings, many promising single-cell experiments deliver biased results or fail to deliver results entirely.
The urgency for technological platforms that are gentle (to minimize cell damage), and efficient (to maximize the capture of all live cells in the population while maintaining the original cellular representation) is imperative to realize the scientific and clinical potential of single-cell analysis.
The LeviCell systems deliver on that promise.
Library preparation
To demonstrate the benefits of the LeviCell systems in removing dead cells and debris, each sample of 400k cells was split into fractions prior to each enrichment process. One half of each sample was washed into a 10x library preparation buffer, while the other half was enriched for viable cells using the LeviCell 1.0 system. After quantitation, the output from the LeviCell 1.0 was put directly into the Chromium Next GEM Single Cell 3’ kit step without additional washing. Approximately 15k cells were used for each method. Final cDNA concentration was estimated by BioAnalyzer. While all samples succeeded at this step, there was a clear improvement in the quality of the cDNA and the resulting library quality from the material generated by the LeviCell.
All samples were normalized before library preparation to account for differences in yield; after normalization, 15K cells were loaded from each process into the Chromium system.
No negative effects on post-processing population representation
The data below show how LeviCell’s gentle cell separation approach enables the study of notoriously delicate and sensitive primary cell types without compromising the integrity of the original cell populations. Sorted and unsorted ovarian DTC samples were blocked with TruStain human Fc Block for 15 minutes at RT then stained with 5 μL each of anti-CD45, anti-CD19, anti-CD11b, and 10 μL of anti-CD3, for 30 minutes at room temperature. Samples were washed with FACS buffer (0.5% BSA in PBS) once before analyzing on a Sony SH800S cytometer. The LeviCell output shows a similar amount of CD3+, CD19+, and CD11b+ cells within the CD45+ lymphocyte population while the sample processed by the bead method shows reduced proportions of CD3+ and CD11b+ cells.
Early stage cancer sample
The distribution of UMI counts per cell in the standard method shows a small cluster of cells with the majority of the UMI counts. This same cluster of cells is represented by a single cluster when grouped by gene expression changes. In contrast, in the LeviCell enrichment sample there is a larger number of cells with significant numbers of UMI counts. This same group of cells is represented by four gene expression clusters (lower right), indicating a larger range of cellular diversity captured in the sequencing results.
t-SNE Projection
Late stage cancer sample
A similar trend is observed with the late stage cancer sample, although the trends are less pronounced. Here, the sample processing with the standard method shows a large number of cells with high diversity of UMI counts, and correspondingly five clusters of gene expression. After enrichment on the LeviCell 1.0 system, the sample has a larger number of cells with high diversity of UMI counts, and six clusters of gene expression clusters, indicating a superior view of the expression profiles.
t-SNE Projection
High quality cells produce the highest quality data
The LeviCell systems’ ability to seamlessly enrich for target cells and produce robust yields of viable cells without preferentially depleting or changing the frequency or expression of cell types gives it the necessary technological characteristics that many scientists have been waiting for to take them to the next level.
Related Documentation
- Single-cell data at scale
Model-ready single-cell data, plate after plate
Cell atlases, perturbation screens and the training sets behind large models of cell biology need thousands of samples prepared exactly the same way. Dead cells and debris lower data quality and inflate cost per usable cell, and manual prep adds run-to-run variability that no downstream analysis can remove.
The LeviCell® 96 System brings label-free Levitation Technology™ to standard SBS-compatible robotics, so sample prep scales with your sequencing capacity instead of your headcount.
Enrich
Remove dead cells and debris before encapsulation.
Wash
Buffer exchange for CITE-seq and other antibody-staining workflows.
Concentrate
Bring cells or nuclei to loading concentration in the buffer of your choice.
Collect
Deliver ready-to-load samples to an output plate for your single-cell platform.
- Cells and nuclei: 15,000 to 500,000 cells per well, plus automated nuclei isolation, wash and elution.
- Unbiased by design: no labels, beads, high pressure or microfluidics, so expression and activation states are preserved.
- Consistent well to well: every sample sees the same protocol, removing manual variability between runs and operators.
- Lower cost per sample: $10 vs >$100, with 96 samples enriched in 45 minutes.