The LeviCell™ System Establishes Highly Effective, Label- and Culture-Free, Robust Bacterial Enrichment, and Separation from Mammalian Cells
Introduction
Early isolation of microbial cells from human samples is essential for positive identification of the pathogen and subsequent development of viable therapies that enhance patient outcomes. Classifying bacteria at or near the point of care allows clinicians to initiate optimal treatment options at the earliest stages of infection. Additionally, microbial identification has proven to be beneficial in the design of effective quality control methods for food and water safety.
Currently, the accepted standard for bacterial characterization is based on phenotypic analysis in cell culture. This method requires 24 hours to a few days between sample collection, culture, analysis, and diagnosis – adding a time challenge to an already complex situation for guiding the initial stages of treatment. The LeviCell significantly reduces this hurdle.
The LeviCell platform consistently demonstrates the effective removal of dead cells from a bacterial population in under one hour, which reduces the number of contaminants in the downstream culture. A pure, viable population maximizes the efficacy of later antibiotic treatment assays. The LeviCell can also be used to remove mammalian cells from a mixed population containing bacteria. In addition, levitation technology has proven effective in separating populations of infected cells from uninfected.
Methods
The LeviCell’s three-step process eliminates the need for cell surface markers and antibodies – which often leads to preferential selection, cell damage, or cell activation. By simplifying the sample preparation procedure, the LeviCell reduces the risk for error while maximizing the potential success of downstream assays that include antibiotic resistance, food quality, water quality, and sepsis therapies.
Results
Separation of Mammalian Cells from Bacteria
The following image shows the LeviCell platform’s demonstrated ability to efficiently and effectively separate bacterial cells from THP-1, a human monocytes cell line. Note the clear line of delineation. The collection of pure and viable E. coli cells for downstream assays increases the chances of successful subsequent analysis by eliminating the background of the mammalian cell population.
Separation of live vs dead Bacteria
In the following experiment, an E. coli sample was treated with gentamicin and mixed with an untreated sample to generate a mixed
population, which was then loaded into the LeviCell. Live and dead populations are clearly observed levitating at different heights. The ability to test responses of different populations of bacteria to a variety of compounds without the need to culture and collect the resulting cells for downstream analysis is especially important when studying populations which are typically difficult – if not impossible – to culture.
Separation of Infected vs Uninfected Macrophages
In this example, the macrophage cell line RAW264.7 was infected with the bacterium Salmonella enterica. After infection, both the uninfected and the infected populations were fixed and loaded into the LeviCell for separation. The bulk of the cells post-infection clearly levitate higher than the uninfected cells, which simplifies the identification and collection of cells for downstream analysis.
Separation of infected vs. uninfected macrophages (Salmonella)
Conclusion
Efficient microbial isolation from a mixed population sample is vital to the identification and characterization of the pathogen as well as the design and development of therapies against it. Common sorting, enrichment, and purification methods typically require hours of labor by a highly skilled technician to generate sufficient yield for downstream applications. Furthermore, current methods necessitate the labeling and culturing of bacteria. The repeated physical manipulation of the cells, which increases the risk of contamination, activation, and cell death. LeviCell offers an immediate solution to these challenges.
By utilizing unique characteristics of target cells to separate them from a mixed population, the LeviCell eliminates the need for labels, culturing, and manual manipulation of bacteria during the enrichment process – thereby increasing purification and yield while significantly decreasing the amount of time spent preparing samples for downstream analysis.
