Tumor Processing

Introduction

The key to unlocking targeted cancer therapies that will improve patient survival lies in the isolation and analysis of live cells in the tumor microenvironment. As primary cells that offer the closest representation of the original cancerous tissue, dissociated tumor cells (DTC) contain tumor, immune, and support cells. Each cell type plays a distinct role in tumorigenesis. Hence, the preservation of each cell type’s population ratio within the microenvironment is vital to the integrity of any downstream application and analysis.

Working with dissociated tumor cell samples is known to be technically challenging, involves rigorous incubation periods, and is associated with low cellular integrity and viability for downstream assay use. Traditionally, the dissociation process is performed using a combination of enzymatic and mechanical separation, which often results in the damage and degradation of cells. Consequently, DTC samples contain a high percentage of debris that interferes with post-processing analysis.

The LeviCellTM removes debris and enriches for live cells while preserving each unique cell type’s population ratio within the original tumor microenvironment. The aggravating issue of contamination is eliminated by having the entire dissociation process occur in the LeviCellTM’s closed, touch-free, self-contained environment. Furthermore, the LeviCellTM’s ability to produce robust, viable cells and abundant yield removes the tedious need for serial subculturing to enrich the tumor cells, thus reducing the overall financial burden and time investment associated with dissociating tumor cells.

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Results

The following results illustrate how the LeviCellTM removes debris as it enriches for live cells while maintaining cell population representation.

The above plots and tables show pre and post LeviCellTM FACS and Cellometer results indicating increased viability in the live channel following sorting with the LeviCellTM. The plots and tables on the left were generated from Endometrial DTCs, while the data on the right is from Bladder DTCs. Samples that were or were not sorted with the LeviCellTM were stained with DAPI and analyzed by flow cytometry and the results are shown in the FACS plots above. Samples were also quantified using a Cellometer results of which are reported in the tables.
The above graphs show pre- and post- LeviCellTM FACS runs showing that unique populations are maintained within the same ratios. The left indicates results from Endometrial DTCs, and the graph on the right results from Bladder DTCs.
This graph illustrates how the LeviCellTM removed a considerable amount of debris from Bladder and Endometrial DTC samples while preserving starting viability (Dark Green), LeviCellTM enriched viability (Light Green).

Dissociated Tumor Cells

Viable DTCs are an integral component of therapeutic, diagnostic, and preclinical cancer research. Current methods used for sorting dissociated tumor cells take a certain level of finesse and technical expertise, time, and ample funding to produce sufficient yield and DTC viabilities that enable the analysis of these rare and sensitive cells.

The LeviCellTM’s 3-step, hands- and label-free platform enables the enrichment of DTCs while maintaining their gene expression and original sample representation ratio for accurate downstream analysis. This unique combination of technological features enables scientists and researchers to focus their effort and expertise on advancing their life-saving scientific knowledge and discoveries.

Processing Macrophages

As scientists and researchers working with macrophages in vitro know all too well, the promise of new and powerful insights in science that these key cell types offer come at a price of time, frustration, and challenges in reproducing results.  Mature differentiated macrophages are present in very low numbers in various human tissues, so it is difficult to have enough quantity for the various downstream assays.  Macrophages cannot proliferate in vitro – they need to be isolated and differentiated with specific media. On top of that, macrophages are notoriously sensitive to handling suing tradiitonal methods and techniques.

The LeviCellTM has proven to successfully sort live macrophages with class-leading yields and viabilities – without affecting activation state – for accurate downstream analysis in 20 touch-free, label-free, frustration-free minutes. Furthermore, result reproducibility is maximized because the entire sorting and separation process occurs in the completely closed and controlled environment of the LeviCellTM and involves only 3 simple manual steps (instead of the complex 13-16  step processes of current methods.

Results

LeviCellTM enriches for the live macrophage cells

The RAW264.7 mouse macrophage cell line was used to determine whether the LeviCellTM was gentle on fragile cells such as macrophages.

Cells were harvested from culture (RAW264.7 cells are cultured in DMEM buffered with 10% FBS) and their viability determined by Trypan blue staining. RAW264.7 macrophages were then washed in a levitation agent and immediately introduced into the LeviCellTM.

The starting viability was measured at 21% (green bar). In the LeviCellTM, live cells levitated to the top channel while the dead cells could be found levitating lower. The LeviCellTM sort (black bar) enriched the Live channel to a viability of 98%!

This demonstrates that not only is the LeviCellTM able to enrich live cells, but it is also gentle on the cells, resulting in very little damage to the cells during the sort.

LeviCellTM maintains macrophages’ polarization state

The RAW264.7 mouse macrophage cell line was used to determine whether the LeviCellTM was gentle on fragile cells such as macrophages.

Cells were harvested from culture (RAW264.7 cells are cultured in DMEM buffered with 10% FBS) and their viability determined by Trypan blue staining. RAW264.7 macrophages were then washed in a levitation agent and immediately introduced into the LeviCellTM.

The starting viability was measured at 21% (green bar). In the LeviCellTM, live cells levitated to the top channel while the dead cells could be found levitating lower. The LeviCellTM sort (black bar) enriched the Live channel to a viability of 98%!

This demonstrates that not only is the LeviCellTM able to enrich live cells, but it is also gentle on the cells, resulting in very little damage to the cells during the sort.

We observed an enrichment to 88-100% viability post-LeviCellTM sort. We took a step further and performed qRT-PCR, looking at the expression of genes indicative of the M1 (iNOS) and M2 (Arg1) states in these cells to show that their activation states were not compromised by the LeviCellTM sort.

No differences in gene expression were observed between the input and output cells for all the activation states. This shows that the LeviCellTM sort is gentle enough to sort macrophages without affecting their polarization states, thus working as a powerful tool for studying the functionality of these cells.

Fine Needle Aspirate

The collection of guided fine needle aspirates (FNA) from tumors is a well-established clinical procedure used to help identify various lymphomas. The integrity of the process is influenced by several factors, including the delicate practice of sample processing for downstream analysis. Since there are no universal guidelines for handling FNA samples, diagnostic accuracy and test sensitivity vary widely among institutions.

Consequently, low cell count, diminished viability, high levels of debris, and population representation stability are constant challenges faced by scientists.

By consistently producing highly viable and replicable results from limited FNA samples, the LeviCell™ provides a cost-effective and time-saving solution that can potentially set the standard for isolating viable cells for FNA sample processing.

Cells are currently used to replace cells damaged by chemotherapy, and researchers are testing ways to incorporate regenerative therapies for cancer, inflammatory diseases, autoimmune disorders, and neurological diseases.

However, successfully culturing stem cells – which are notoriously sensitive – pose challenges that can result in significant additional costs and delays. Integrating the LeviCell™ platform into stem cell research laboratories as a tool for safely and effectively isolating differentiated cells from culture will enable scientists to generate consistent and reproducible results at a lower cost and shorter timeline than traditional methods.

Methods

As the world’s first label-free cell separation technology, the LeviCell leverages the size and density of cells to isolate them from other cell types.  The LeviCellTM gently displaces target cells using less than 0.5 psi of force, which has proven to have no negative effects on even the most sensitive cell types. In comparison, conventional methods exert 70 – 100 psi of force, which damages hearty cells and completely destroys sensitive ones. The LeviCellTM is the only label-free technology in the market that can isolate and enrich a robust quantity of viable cells from high-value primary samples and sensitive cell types without affecting original population representation.

Using the LeviCellTM involves three easy manual steps. First, pipette the FNA sample directly from the collection tube into the single-use, biocompatible, sterile consumable.  Second, load the cartridge into the LeviCellTM platform. The LeviCellTM automatically processes the sample and separates live cells from dead, which will levitate at a lower height and is collected in the bottom channel of the consumable cartridge.  Live cells will be collected in the top channel.  This process will take approximately 30 minutes to complete.  Upon completion, collect target cells for downstream analysis, which is the third step of the LeviCellTM cell separation workflow.  Simple. Quick. Easy.

STEP 1

Sample Introduction

Sample pipetted into the LeviCell™ cartridge.

STEP 2

Automated Label-Free Sorting

Precision density gradients drive levitation based on cells physical properties.

STEP 3

Sample Collection

Selected populations collected into separate ports for further analysis or use.

A five-fold increase in Viability

FNA samples were collected from human H358 tumor xenografts in rats by Hera Biolabs. An FNA sample was suspended in Levitation BufferTM and enriched for live cells using the LeviCellTM. A portion of the sample was set aside. The live channel output and the unsorted sample were stained with Trypan and counted on a microscope. The LeviCellTM output shows a nearly 5-fold increase in viability compared to the input.

Debris-Free Sample

FNA samples from before and after sorting on the LeviCell were stained with Trypan and counted on a microscope. The images show a reduction in the number of dead cells and debris after sorting with the LeviCell.

No negative effects on population representation

To demonstrate the LeviCellTM’s gentle process – which enables robust yields without affecting population representation of the original sample – PBMCs were suspended in Levitation AgentTM and enriched for live cells using the LeviCellTM. A portion of the original sample was analyzed independently. Both live and dead channel outputs as well as the unsorted sample were blocked with TruStain human Fc Block for 15 minutes at RT then stained with 5 μLeach of anti-CD45 (PE), anti-CD3 (FITC), anti-CD11b (APC), and 10 μL of anti-CD19 (PerCP-Cy5.5) for 1 hour on ice. Samples were then washed with a FACS buffer (0.5% BSA in PBS) once prior to analysis on a Sony SH800S cytometer.

The live channel output (red bar) shows a similar amount of CD3+, CD19+, and CD11b+ cells within the CD45+ lymphocyte population as was present prior to sorting (blue bar). The consistency of robust live cell yield across all cell types is testament to how the LeviCellTM platform’s fast, gentle, and powerful separation technology enables the preservation of original population representation.

The LeviCellTM is uniquely positioned to transform the FNA sample processing workflow by delivering consistently robust and replicable results from extremely sensitive and limited FNA samples, without negatively affecting original population representation.  The revolutionary cell separation technology negates the need for markers, manual handling, and exertion of force greater than 1 psi – any of which can negatively affect cells and influence downstream results.  This is a significant development in the field, as FNA samples are difficult to procure, challenging to work with, and hard to replace. The LeviCellTM not only evens out the playing field when it comes to FNA sample processing, it completely changes the game by setting the standard.

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Live Cell Enrichment