Single Cell Sequencing

Introduction

Single-cell analysis technologies have been instrumental in facilitating significant developments in cellular biology. Rapid progress in this growing field is necessary for the continued transformation of how we learn about and approach the clinical research, diagnosis, prognosis, and treatment of a wide range of disease types. However, the more we learn about the immense heterogeneity of cell populations, the more we realize that bulk population analysis and current tools for preparing samples for single-cell analysis damage or negatively affect cells during sample preparation, and do not provide adequate representation of the true cell population within the sample.

Conventional single-cell sample preparation methods and technologies tend to damage cells, have low capture efficiency, and generally result in low cell viability – resulting in inaccurate population representation of the biological sample or insufficient cells for a complete experiment. Often, scientists discover these issues late in the experimental pipeline, which contributes to their increasing frustration and costs in irreplaceable time, resources, and funds. 

The urgency for technological platforms that are gentle (to minimize cell damage) and efficient (to maximize the capture of all live-cell types in the population) is imperative to realize the scientific and clinical potential of single-cell analysis.  The LeviCell™ demonstrates such a promise.

Methods

Levitas Bio has developed an innovative levitation technology platform that uses less than 1 psi of pressure to enable a completely touch-free, label-free, three-step sorting process that takes only 20 minutes to complete.

Live cell populations levitate much higher than dead cells and debris, allowing for clear separation and collection by the LeviCell™ , as shown.  Please note that the fluorescent dyes used in the provided image were used solely to highlight the separation process and are not required to use LeviCell™. 

LeviCell’s™ gentle separation process maintains maximum cell viability, enabling the preservation of cell population representation integrity for downstream assays such as single-cell analysis.  The simple three-step process streamlines the workflow and minimizes the risk of contamination and damage from multiple instances of handling and washing, consequently maximizing enrichment for each cell type. 

Results

Primary samples were analyzed on a Sony® SH800S cell sorting instrument before and after enrichment with the LeviCell in order to validate the performance of the system. 

Revolutionary viability and yields

LeviCell™  demonstrated the successful removal of dead cells and debris from a variety of primary samples while maintaining the original representation of the populations found within the sample. Furthermore, LeviCell™ was able to enrich a low starting number of live cells to a viability of >95%, exhibiting a capacity for scalability.

Ethanol-killed Jurkat cells were mixed with fresh live Jurkat cells to obtain a 20-25% viable mixed population.

A total of 200K cells was enriched for live cells across different systems to determine purity and yield.

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.

PBMCs were suspended in levitation agent and enriched for live cells using the Levicell. A portion of the sample was set aside. Both live and dead channel outputs and the unsorted sample were blocked with TruStain human Fc Block (CAT#) for 15 minutes at RT then stained with 5 μL each of anti-CD45 (PE, CAT#), anti-CD3 (FITC, CAT#), anti-CD11b (APC, CAT#), and 10 μL of anti-CD19,  (PerCP-Cy5.5, CAT#) for 1 hour on ice. Samples were washed with FACS buffer (0.5% BSA in PBS) once before analyzing on a Sony SH800S cytometer. The live channel output shows a similar amount of CD3+, CD19+, and CD11b+ cells within the CD45+ lymphocyte population.

No effect on the post-processing activation state

Data exhibited below clearly illustrates that LeviCell™ has no effects on the post-processing activation state of cells.

We performed qRT-PCR, looking at the expression of M1 (iNOS) and M2 (Arg1) in these cells to show that their activation states were not compromised by the LeviCell sort. No differences in gene expression were observed between the input and output cells for all the activation states.

Conclusion

The innovative label-free separation technology of the LeviCell facilitates complete debris and dead cell removal without affecting the original population representation of gene expression. 

Additionally, LeviCell’s quick and gentle sorting ensures robust viability and high live-cell yield, which reinforces the ability to produce the most biologically relevant results. Because the separation process takes only 20 minutes, hands-on time and the number of manual steps are reduced by more than 80% in comparison to traditional methods, thus minimizing the amount of time and effort spent on sample preparation while maximizing the production of the most viable cells possible.