A 3D in vitro assay to study combined immune cell infiltration and cytotoxicity.

Immune cell-mediated killing of cancer cells in a solid tumor is prefaced by a multi-step infiltration cascade of invasion, directed migration, and cytotoxic activities. In particular, immune cells must invade and migrate through a series of different extracellular matrix (ECM) boundaries and domains before reaching and killing their target tumor cells. These infiltration events are a central challenge to the clinical success of CAR T cells against solid tumors. The current standard in vitro cell killing assays measure cell cytotoxicity in an obstacle-free, two-dimensional (2D) microenvironment, which precludes the study of 3D immune cell-ECM interactions. Here, we present a 3D combined infiltration/cytotoxicity assay based on an oil-in-water microtechnology. This assay measures stromal invasion following extravasation, migration through the stromal matrix, and invasion of the solid tumor in addition to cell killing. We compare this 3D cytotoxicity assay to the benchmark 2D assay through tumor assembloid cocultures with immune cells and engineered immune cells. This assay is amenable to an array of imaging techniques, which allows direct observation and quantification of each stage of infiltration in different immune and oncological contexts. We establish the 3D infiltration/cytotoxicity assay as an important tool for the mechanistic study of immune cell interactions with the tumor microenvironment.

Enhanced filtration performance using feed-and-bleed configuration for purification of antibody precipitates.

Precipitation can be used for the initial purification of monoclonal antibodies (mAbs), with the soluble host cell proteins removed in the permeate by tangential flow microfiltration. The objective of this study was to examine the use of a feed-and-bleed configuration to increase the effective conversion (ratio of permeate to feed flow rates) in the hollow fiber module to enable more effective washing of the precipitate. Experiments were performed using human serum Immunoglobulin G (IgG) precipitates formed with 10 mM zinc chloride and 7 wt% polyethylene glycol. The critical flux was evaluated as a function of the shear rate and IgG concentration, with the resulting correlation used to predict conditions that can achieve 90% conversion in a single pass with minimal fouling. Experimental data for both the start-up and steady-state performance are in good agreement with model calculations. These results were used to analyze the performance of an enhanced continuous precipitation-microfiltration process using the feed-and-bleed configuration for the initial capture / purification of a mAb product.