Tissue Processing
The LeviCell™ Demonstrates Fast and Robust Live Cell Enrichment from a Range of Tissue Types.
Introduction
Processing tissue samples, such as resections and biopsies, is one of the most vital steps in sample preparation, and also the most challenging. Since these samples are often used for research and diagnostic purposes, it is critical to maintain cell population representation ratios from the original sample while ensuring successful enrichment of target cells.
The typical enrichment and debris removal processes require multiple complex steps that include exposing the sensitive cells to harsh physical and chemical conditions, resulting in low viability and yield, which are a common source of frustration for many scientists and researchers.
Requiring only 3 steps and less than 20 minutes, the LeviCell system harnesses the power of levitation technology to efficiently remove dead cells and debris while delivering enriched cells from a variety of tissues with consistently higher yields and viabilities, and without alterations to their gene expression profile. With the LeviCell system, you can now gently select for viable cells from tissues without the need for cell surface markers or antibodies, which often lead to biased selection, cell activation, and cell death.
Quick links to sections
Dissociated Tumor Cells
Cardiomyocytes
Alveolar Type II (ATII) Cells
Adipocytes
3-step LeviCell Workflow
Dissociated Tumor Cells
Comparison of Two Enrichment Techniques Using Three Cancer Cell Types
Tumor cells from bladder, colorectal carcinoma, and renal cell carcinoma were processed and enriched with two different methods, a magnetic bead-based protocol, and the LeviCell system which uses levitation technology.
Frozen dissociated tumor cells were thawed at 37ᵒC. With a small amount of ice remaining in the vial, the thawed cells were transferred into 10 mL of warmed media + 10% FBS dropwise. Cells were centrifuged at 500 rcf for 5 minutes. The supernatant was removed, and the pellet was resuspended in 10 mL of warm media. Cell count and viability were determined via Trypan blue staining at a 1:1 dilution.
The following graph compares starting (blue) and final (green) viabilities of bladder tumor, colorectal carcinoma, and renal cell carcinoma cells using bead-based enrichment and the LeviCell platform. The LeviCell’s superior technical ability to enrich different cell types is evident in the results.
LeviCell Enrichment Delivers Higher Live Cell Yield Than Bead-Based Enrichment
Levitation Technology Preserves Original Population Representation
Non-small Cell Lung Carcinoma (NSCLC) was enriched for high viability using the LeviCell from a starting viability of 58% to a final viability of 79% (A). The live channel output (red box) shows a similar amount of CD45+, as well as CD3+, CD19+, and CD11b+ cells within the CD45+ lymphocyte population as was present prior to sorting (blue bar) (B). The consistency of robust live cell yield across all cell types is testament to how the LeviCell platform’s fast, gentle, and powerful separation technology enables the preservation of original population representation.
Unmatched Debris Removal
In addition to viable cell enrichment, the LeviCell system enables simultaneous removal of dead cells and debris. In this set of images, the first one depicts the original debris observed in the sample. The live channel, where the viable cell population is found, is debris-free, while debris is seen in the dead channel (Image 3).
LeviCell Accommodates a Variety of Tissue Type
The LeviCell System can process a variety of sample and cell types, including bladder tumor, colorectal carcinoma, glioblastoma, lung, melanoma, non-small cell lung cancer (NSCLC), pancreas, peripheral blood mononuclear cells (PBMCs), prostate tumor, and renal cell carcinoma. See the full list of cells and sample types.
Cardiomyocytes
With heart disease being one of the leading causes of death worldwide, deeper insight into the possible roles that cardiomyocytes play in the regeneration and repair of the heart is key to understanding the pathogenesis of cardiovascular disease, which may lead to the development of life-saving therapies. While surgical intervention (bypass, catheter, assist devices) continue to be the most widely implemented treatment, the nature of cardiovascular disease inevitably leads to heart failure – and subsequent need for a heart transplant.
Since the demand for heart transplantation far exceeds the available supply, cell-based cardiac repair therapies are being aggressively pursued as a more pragmatic, cost-effective, and low-risk option. However, current marker-based selection methods have been ineffective in the isolation and enrichment of cardiomyocytes, which remain as one of the most challenging cell types to process due to their sensitivity and fragility.
Offering a marker-free approach, the LeviCell system successfully enriches differentiated cardiomyocytes without activating the cells or affecting population representation, enabling research that may lead to future development of novel therapies for cardiovascular disease.
Differentiated vs. Non-Differentiated Induced Pluripotent Stem Cells
Induced pluripotent stem cells (iPSCs) were differentiated into cardiomyocytes and metabolically starved before resuspending in levitation agent. Cardiomyocytes were enriched from the undifferentiated iPSCs and fibroblasts by processing with the LeviCell. Outputs were cultured in 6 well plates for 3-5 days to determine phenotype and viability. The separation on the LeviCell can be seen below in Fig. A
The density and resulting levitation height of the differentiated cardiomyoctyes is significantly higher than the non-cardiomyocyte population and can be clearly seen in the density graph Fig. B and the enriched population vs. un-enriched in Fig. C.
Alveolar Type II (ATII)
During the current COVID-19 pandemic, the need to understand the pathology of SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2), the virus (responsible for COVID-19, is immediate and critical to the development of vaccines and life-saving therapeutics. One of the primary targets of the SARS-CoV-2 is the alveolar type II (ATII) cell in bronchial tissue. ATII cells, typically found at the blood-air barrier, are involved in the innate immune response mechanism and play an instrumental role in the replacement of ATI cells, which are responsible for gas exchange between alveoli and blood. They also secrete pulmonary surfactant which prevents collapse of the alveoli, known as atelectasis.
Despite their key roles, ATII cells are challenging to isolate and study due to their tendency to differentiate into alveolar type I (ATI) cells in culture and their susceptibility to fibroblast contamination from primary isolation. Commercially available methods were not able to effectively isolate ATII cells from primary tissue, and published cases of successful ATII isolation indicated substantial investments in equipment and highly specialized scientists were required. This adds a layer of complexity, cost, and time needed for improvement and optimization of cellular assays, thus delaying the drug development process. For this reason, most therapeutics in development for COVID-19 relief efforts employ standard immortalized cell lines that are easy to handle and provide longevity, but may not retain biological relevance for the actual cellular systems affected by SARS-CoV-2.
In contrast, primary cells such as ATII cells derived from human tissue do represent a physiologically relevant system suitable for assessment and optimization of cellular assays that target SARS-CoV-2, and may inform future development of treatments and therapies. The ability to isolate and enrich primary ATII cells, is therefore paramount.
Current isolation and enrichment techniques rely on labeling, which creates downstream challenges such as decreased viability of sensitive cell types; isolates with unsuitable purity for culture; a need for large initial cell population; high cell death rate; accidental transformation; and potential aerosol generation during the separation process. This last issue is particularly problematic with SARS-CoV-2, as aerosolization of virus particles places scientists at higher risk for exposure and contracting of the virus.
The LeviCell system successful separates cell lines from primary human tissues with a cost-effective, simple, timesaving, easily replicable and aerosol-free method. Isolation of infected and non-infected ATII cells from human tissue can be achieved using the LeviCell without sacrificing viability, enrichment, or assay integrity, enabling us to support the broader SARS-CoV-2 research efforts.
Methods
To meet the need for high-viability isolation of sensitive and difficult cell types such as ATII cells, LevitasBio developed the LeviCell system – a powerful, label-free cell sorting platform to gently, rapidly, and efficiently separate and enrich cells. With its innovative magnetic levitation technology that exploits the intrinsic density and magnetic signature differences of cells, the LeviCell improves upon current cell separation methods in three ways.
First, the LeviCell processes cells without the use of labels, the use of which can activate sensitive cell types such as macrophages, adipocytes, cardiomyocytes, and ATII cells. Second, because the LeviCell platform’s process is fast, simple, and reliable, it provides a reproducible method of cell isolation that does not require expensive and complicated workflows. Third, the LeviCell separation system can process typical and difficult-to-isolate cell types – at any starting cell population number – giving it broad applicability that no other existing cell separation technology has.
These qualities make the LeviCell an ideal technology to rapidly address the technical challenge of efficiently and consistently separating primary ATII cells, while generating high viability from human samples for urgent use in COVID-19 therapeutic research and development.
Conclusion
The LeviCell platform has demonstrated the separation of many cell lines that include highly sensitive cell types (such as ATII cells) that are not easily isolated using conventional methods.
Current techniques rely on labeling, which frequently poses issues such as the reduction in the viability of sensitive cell types; isolates with unsuitable purity for culture; a necessity for large initial cell population; high cell death rate; accidental transformation; and potential aerosol generation during the separation process. This last issue is particularly problematic with SARS-CoV-2, as aerosolization of virus particles places scientists at higher risk for contracting the virus. The LeviCell offers a cost-effective, simple, timesaving, easily replicable, and aerosol-free method of isolating infected and non-infected ATII cells from human tissue without sacrificing viability, enrichment, or assay integrity.
The distribution of the LeviCell into laboratories will enable the acceleration of COVID-19 therapeutics development and support the broader SARS-CoV-2 research effort.
Results
Brightfield (left) and fluorescence (right) microscopy images of ATII cell separation on the LeviCell system. ATII cells were labeled with HTII-280 and are visualized while still in the separation channel. The reference line indicates the level at which the downstream sample splitter is located. Cells are separated by the splitter and flow to different outlets: Live (top) and Debris/Dead (bottom).
After equilibration, the top and bottom bands (above and below the reference line, respectively) are separated by elution through two different outlets and are imaged in the following figure. Cells are stained with DAPI, anti-KRT5 antibody, and previously applied anti-HTII-280 antibody to ease identification and assess enrichment. DAPI binds AT-rich regions of DNA, indicating the location of cellular nuclei, while KRT5 is a defining marker for pulmonary basal cells.
Images of cells after LeviCell separation. Top Band (top panels) show samples enriched for ATII cells stained red with HTII-280 and Bottom Band (bottom panels) show residual cells present in the sample.
Images of separated cells show significant enrichment for ATII cells in top panels by the elimination of a significant proportion of alveolar type I cells and other cells compared to the sample collected from the bottom band. These data strongly support the LeviCell platform’s ability to deliver an optimized protocol for separation and enrichment of ATII cells that are highly viable and suitable for culture.
Adipocytes
The alarming rate at which obesity is growing worldwide has intensified the necessity and urgency of obtaining a deeper understanding of all aspects of adipocyte biology, as obesity often leads to numerous chronic and costly health conditions. Substantial discoveries have been made in the complex functions of adipocytes that have led to advances in endocrinology, cardiology, energy metabolism, cancer biology, and developmental and stem cell biology. Scientists continue to study the implications of managing adipocyte growth and proliferation in a variety of disease processes to uncover opportunities for unique therapeutic interventions. The ongoing quest for insight into the varied roles and mechanisms of these cell types puts adipocyte research at the heart of a variety of diseases such as diabetes, hypertension, heart disease, metabolic syndrome, sleep disorders, and cancer. As such, the need to isolate and characterize adipocytes is immense. However, adipocytes are notoriously problematic to work with. These highly sensitive large cells tend to form bulky clumps that make them challenging to isolate using conventional separation techniques such as FACS. The degree of differentiation in precursor models also poses considerable challenges in assessment and downstream applications due to the limitations in current techniques that leave an abundance of undifferentiated cells in the samples. The LeviCell provides a solution to these limitations. The LeviCell platform separates cells based on physical properties such as density. Adipocytes have lower density compared to most mammalian cells due to their lipid-rich vesicles causing them to levitate higher in the LeviCell environment. The nature of the LeviCell technology enables robust enrichment, label-free isolation, and minimal cell loss and damage of these delicate adipocytes. Since the entire process occurs in a completely closed environment, prep time, contamination potential, and cellular damage from repeated handling are minimized. Furthermore, the gentle processing method of the LeviCell, which uses <1 psi of pressure, accommodates the safe and effective isolation of large cell clumps (up to 400µm) – ensuring maximum viability, even with these highly sensitive cells.
Results
Cell Differentiation
Cell Preparation for LeviCellTM
qRT-PCR
Conclusion
The LeviCell‘s demonstrated ability to efficiently and effectively isolate large, fragile adipocytes in a gentle, label-free, closed environment shows the platform’s immense potential to reset the bar in cell separation technology. Significant advances in understanding adipogenesis, adipocyte-related diseases, and fat tissue engineering are within reach as the LeviCell unambiguously exhibits its ability to enrich an abundance of pure and viable adipocytes in a shorter time span than conventional techniques.
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Live Cell Enrichment from a Range of Tissue Types
Marker Free Enrichment of Cardiomyocytes
How the LeviCell System Can Help Fight COVID-19
Enrichment Pure Populations of Adipocytes in Differentiated Precursor Cell Models