Non-invasive image-based cytometry for high throughput NK cell cytolysis analysis.

Natural Killer (NK) cells are lymphocytes that are the first line of defense against malignantly transformed cells, virally infected cells and other stressed cell types. To study the cytolytic function of NK cells in vitro, a cytotoxicity assay is normally conducted against a target cancerous cell line. Current assay methods are typically performed in mixed 2D cocultures with destructive endpoints and low throughput, thereby limiting the scale, time-resolution, and relevance of the assay to in vivo conditions. Here, we evaluated a novel, non-invasive, quantitative image-based cytometry (qIBC) assay for detection of NK-mediated killing of target cells in 2D and 3D environments in vitro and compared its performance to two common flow cytometry- and fluorescence-based cytotoxicity assays. Similar to the other methods evaluated, the qIBC assay allowed for reproducible detection of target cell killing across a range of effector-to-target ratios with reduced variability. The qIBC assay also allowed for detection of NK cytolysis in 3D spheroids, which enabled scalable measurements of cell cytotoxicity in 3D models. Our findings suggest that quantitative image-based cytometry would be suitable for rapid, high-throughput screening of NK cytolysis in vitro, including in quasi-3D structures that model tissue environments in vivo.

Real-time transfer of lentiviral particles by producer cells using an engineered coculture system.

Lentiviruses are quite effective gene delivery systems for stable production of genetically engineered human cells. However, prior to using lentivirus to deliver genetic materials to cells of interest, the normal course of production of these lentiviruses involves a lengthy collection, purification, preservation, and quantification process. In this report, we demonstrate the ability for producer HEK293T cells to simultaneously produce lentiviral particles and transduce (i.e., infect) target cells through a membrane-based coculture system in a continuous, real-time mode which negates the need for a separate viral collection and quantification process. The coculture system was evaluated for major design features such as variations in HEK293T seeding density, target cell type densities, as well as membrane porosities to identify key relationships between lentiviral particle production rate and infection kinetics for adherent and suspension cell types. As a proof-of-concept for the creation of an engineered cell immunotherapy, we describe the ability to engineer human T cells isolated from PBMCs under the control of this coculture system in under 6 days with a GFP construct. These studies suggest the capability to combine and more closely automate the transfection/transduction process in order to facilitate well-timed and cost-effective transduction of target cell types. These experiments provide novel insight into the forthcoming transition into improved manufacturing systems for viral production and subsequent cell engineering.