How the LeviCell™ Can Help Fight COVID-19
During the current COVID-19 pandemic, the need to understand the pathology of the SARS-CoV-2 (Severe Acute Respiratory Syndrome Coronavirus 2) 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, secrete pulmonary surfactant. Without this coating, the alveoli collapse, resulting in atelectasis. ATII cells are also 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.
Despite the ATII cells’ key roles, it is one of the more difficult cell types to isolate and study. This is due in part to the ATII cells’ tendency to differentiate into ATI cells in culture and their susceptibility to fibroblast contamination from primary isolation. Consequently, there are no commercially available methods to effectively isolate ATII cells from primary tissue. Published successful isolation of ATII cells requires highly specialized scientists and equipment, which adds a layer of complexity, cost, and time to the improvement of cellular assays, thus delaying the rate of drug development. For this reason, most therapeutics in development for the COVID-19 relief effort employ standard immortalized cell lines that are easy to handle and long-lasting, but may not retain biological relevance for the actual cellular systems affected by SARS-CoV-2. Primary cells derived from human tissue represent a physiologically relevant system for improving the utility and relevance of cellular assays that would improve pharmaceutical development for the treatment of COVID-19. This is where the LeviCell can contribute.
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.
Healthy human lung samples were labeled with HTII-280, a monoclonal antibody specific to an apical surface membrane protein in ATII cells, to aid in their identification upon levitation in the LeviCell system. The following images show HTII-280 stained ATII cells introduced into the LeviCell, using brightfield and fluorescent microscopy. The majority of ATII cells are found levitating above the reference line, indicating robust viability. Cells below the reference line are likely to be dead, dying, or may represent a different ATII cell subtype.
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.
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.