In cancer research and personalized medicine, new tissue culture models are needed to better predict the response of patients to therapies. With a concern for the small volume of tissue typically obtained through a biopsy, we describe a method to reproducibly section live tumor tissue to submillimeter sizes. These micro-dissected tissues (MDTs) share with spheroids the advantages of being easily manipulated on-chip and kept alive for periods extending over one week, while being biologically relevant for numerous assays. At dimensions below ~420 μm in diameter, as suggested by a simple metabolite transport model and confirmed experimentally, continuous perfusion is not required to keep samples alive, considerably simplifying the technical challenges. For the long-term culture of MDTs, we describe a simple microfluidic platform that can reliably trap samples in a low shear stress environment. We report the analysis of MDT viability for eight different types of tissues (four mouse xenografts derived from human cancer cell lines, three from ovarian and prostate cancer patients, and one from a patient with benign prostatic hyperplasia) analyzed by both confocal microscopy and flow cytometry over an 8-day incubation period. Finally, we provide a proof of principle for chemosensitivity testing of human tissue from a cancer patient performed using the described MDT chip method. This technology has the potential to improve treatment success rates by identifying potential responders earlier during the course of treatment and providing opportunities for direct drug testing on patient tissues in early drug development stages.

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Neutrophils are known to rapidly migrate to sites of infection and injury, and track bacteria guided by spatiotemporally controlled chemokine gradients. Previous studies of neutrophil chemotaxis, using micropipettes and lately microfluidic devices, are limited to stationary sources and gradients. Thus, despite the well‐known ability of neutrophils to track bacteria in vitro, their response to defined moving gradients remains unknown. Here, a “floating” concentration gradient of interleukin‐8 is generated using a microfluidic quadrupole, and neutrophils cultured in a Petri dish are challenged with steep stationary and moving gradients. Individual neutrophils are tracked by live microscopy and their chemotaxis is analyzed. Interestingly, neutrophils are shown to enter the gradient region in a rolling‐like behavior, rapidly adhere to the bare dish, and polarize within 30 s, faster than what has been observed to date. Under stationary gradients, neutrophil migration length is maximal for cells located at the low end of the gradient, whereas under moving gradients, neutrophils migrate over longer distances and the length travelled is independent of their starting position. Furthermore, neutrophils are shown to initiate their migration at a maximum speed, slowing down when migrating deeper into the gradient and eventually stopping. This work lays the foundation for future chemotaxis assays with moving gradients.

Chitosan-based polyplexes are known to traffic through lysosomes for a relatively long time, independent of the degree of deacetylation (DDA) and the number average molecular weight (Mn) of the polymer, even though both of these parameters have profound effects on polyplex stability and transfection efficiency. A better understanding of the lysosomal barrier is paramount to the rational design of vectors capable of overcoming obstacles to transgene expression. The aim of the present study was to investigate if lysosomal transit affects chitosan-based polyplex transfection efficiency in a structure-dependent (DDA, Mn) manner. Toward this end, we analyzed the effects of intracellular trafficking modifying agents on transfection efficiency and intracellular vesicular trafficking of polyplexes with different structural properties and stabilities or nucleic acid binding affinity. The use of agents that modify endosome/lysosome acidification and transit processes by distinct mechanisms and their effect on cell viability, polyplex uptake, vesicular trafficking, and transfection efficiency revealed novel and strong chitosan structure-dependent consequences of lysosomal transit. Inhibiting lysosomal transit using chloroquine significantly increased the efficiency of unstable polyplexes, while having minimal effects for polyplexes with intermediate or high stability. In parallel, specifically inhibiting the acidification of vesicles abrogated transfection for all formulations, suggesting that vesicular acidification is essential to promote transfection, most probably by facilitating lysosomal escape. These results provide novel insights into the structure-performance relationship of chitosan-based gene delivery systems.