James Freyer, CSO
Flow cytometry has been the standard for cellular analysis. However, traditional flow cytometers have technical constraints: single analysis stream; analysis rates <50K particles/sec; sample rates of <250 μL/min; and >50-fold sample dilution with sheath fluid. These issues make current flow cytometers impractical for rare cell analysis, large dilute samples, rapid analysis of blood, or sample reanalysis.
BennuBio’s Velocyt is a parallel flow cytometer that directly addresses these cell analysis pain points by using standing acoustic waves, rather than sheath fluid, to focus a sample directly into 10 parallel particle streams without sample dilution. These streams intersect line-focused lasers, and these intersections are imaged using a high-speed camera. Software converts the images into flow cytometry data. The Velocyt design simplifies workflows by eliminating washing, lysing, and concentration steps, and analyzes samples at 10 mL/min and >100K events/sec. Because the sample is returned undiluted after analysis, the Velocyt offers the novel ability to perform kinetics.
The Velocyt can analyze particles from 1µ to 1mm in diameter. BennuBio CTO Dr. James Freyer is leading the effort to use the Velocyt for drug screening using 3D tissue models that replicate drug effects in vivo, cellular physiology, cell-cell interactions and drug transport.
Spheroids, produced from a mixture of different cells, mimicking a body organ, play an increasingly critical role in cancer research, stem cell biology and drug screening. “Spheroids are a drug screening system that fills the gap between single cells and animal models, potentially reducing use of animals in drug development,” says Dr. Freyer. However, use of spheroids for drug screening has been stymied by lack of techniques for rapidly analyzing spheroid responses to drugs. Current systems use microscopy of spheroids in stationary culture. This is low throughput (<1 spheroid analyzed per second) and maintaining spheroids in stationary culture produces unpredictable and time-dependent gradients in inter-spheroid nutrients, waste products, cellular physiology, and viability as well as inconsistent drug concentrations in and around the spheroids.
The BennuBio Velocyt is the backbone of a high-throughput spheroid screening platform (HTSSP) that directly addresses these limitations in spheroid drug screening. The Company was awarded an $1M Small Business Innovation Research (SBIR) grant from the National Institute of General Medical Sciences to develop the HTSSP. Modifications to the Velocyt flow cell and analysis software will be integrated with a spheroid sample handling system.
Cell-cell interactions are involved in essentially all diseases, so screening drugs on the basis of enhancing or abrogating a specific cell-cell interaction would be a unique and powerful tool
The HTSSP will maintain spheroids in a stirred suspension prior to and after analysis, providing reproducible spheroids and consistent drug exposures. The spheroids are gently pushed through the flow cell for analysis and returned, undiluted, to a second chamber for further culture or reanalysis. Dr. Freyer notes “The HTSSP provides the unique ability to perform drug screening based on kinetic analyses of drug effects in a 3D tissue model.”
In addition to the critical advantages in spheroid sample handling, the HTSSP will also provide unique advantages for spheroid analysis. Whole spheroid parameters (e.g. size, cell count, morphology, apoptosis, viability) can be rapidly measured on thousands of spheroids. According to Dr. Freyer, analysis of a single spheroid sample as a function of time after adding a drug provides a method for quantifying penetration and binding/uptake of drugs in spheroids. These are critical parameters determining drug effectiveness in vivo that are not currently addressed by any screening system. In addition, whole spheroid analysis allows rapid measurement of the composition of different cell types in a co-cultured spheroid. This allows screening assays that target a specific cell-cell interaction, currently not possible with existing technology. According to Dr. Freyer, “Cell-cell interactions are involved in essentially all diseases, so screening drugs on the basis of enhancing or abrogating a specific cell-cell interaction would be a unique and powerful tool.”
Velocyt’s novel optical analysis enables measurement of inter-spheroid fluorescence distributions. Currently, this is done using very slow confocal microscopy. As spheroids transit Velocyt’s line-focused laser, a 2D image of each spheroid in each stream is produced. Since spheroids are, indeed, spherically symmetric, these 2D images represent the spatial distribution of fluorescence within the 3D structure. This innovative analysis approach provides the opportunity to develop more sophisticated drug screening assays that involve measuring distributions of fluorescence within the spheroid structure. “This will allow screening based on drug penetration, spatial variations in viability/apoptosis/necrosis, spatial distributions of cell types and distributions of microenvironmental parameters (e.g. proliferation, metabolic activity, hypoxia), again not possible in current flow cytometers,” states Dr. Freyer.
Established in 2018, BennuBio recently secured a round B funding of $5 million. The one-laser Velocyt model V1 is currently for sale by BennuBio, with a two-laser model expected to reach market Q2 2022. Beta versions of the HTSSP will be available late 2022.