Tissues are complex mixtures of different cell subtypes, and this diversity is increasingly characterized using high-throughput single cell analysis methods. However, these efforts are hindered, as tissues must first be dissociated into single cell suspensions using methods that are often inefficient, labor-intensive, highly variable, and potentially biased towards certain cell subtypes. Here, we present a microfluidic platform consisting of three tissue processing technologies that combine tissue digestion, disaggregation, and filtration. The platform is evaluated using a diverse array of tissues. For kidney and mammary tumor, microfluidic processing produces 2.5-fold more single cells. Single cell RNA sequencing further reveals that endothelial cells, fibroblasts, and basal epithelium are enriched without affecting stress response. For liver and heart, processing time is dramatically reduced. We also demonstrate that recovery of cells from the system at periodic intervals during processing increases hepatocyte and cardiomyocyte numbers, as well as increases reproducibility from batch-to-batch for all tissues.

Tumors tissues house a diverse array of cell types, requiring powerful cell-based analysis methods to characterize cellular heterogeneity and identify rare cells. Tumor tissue is dissociated into single cells by treatment with proteolytic enzymes, followed by mechanical disruption using vortexing or pipetting. These procedures can be incomplete and require significant time, and the latter mechanical treatments are poorly defined and controlled. Here, we present a novel microfluidic device to improve mechanical dissociation of digested tissue and cell aggregates into single cells. The device design includes a network of branching channels that range in size from millimeters down to hundreds of microns. The channels also contain flow constrictions that generate well-defined regions of high shear force, which we refer to as “hydrodynamic micro-scalpels”, to progressively disaggregate tissue fragments and clusters into single cells. We show using in vitro cancer cell models that the microfluidic device significantly enhances cell recovery in comparison to mechanical disruption by pipetting and vortexing after digestion with trypsin or incubation with EDTA. Notably, the device enabled superior results to be obtained after shorter proteolytic digestion times, resulting in fully viable cells in less than ten minutes. The device could also be operated under enzyme-free conditions that could better maintain expression of certain surface markers. The microfluidic format is advantageous because it enables application of well-defined mechanical forces and rapid processing times. Furthermore, it may be possible to directly integrate downstream processing and detection operations to create integrated cell-based analysis platforms. The enhanced capabilities enabled by our novel device may help promote applications of single cell detection and purification techniques to tumor tissue specimens, advancing the current understanding of cancer biology and enabling molecular diagnostics in clinical settings.

Nanoparticles have drawn intense interest as delivery agents for diagnosing and treating various cancers. Much of the early success was driven by passive targeting mechanisms such as the enhanced permeability and retention (EPR) effect, but this has failed to lead to the expected clinical successes. Active targeting involves binding interactions between the nanoparticle and cancer cells, which promotes tumor cell-specific accumulation and internalization. Furthermore, nanoparticles are large enough to facilitate multiple bond formation, which can improve adhesive properties substantially in comparison to the single bond case. While multivalent binding is universally believed to be an attribute of nanoparticles, it is a complex process that is still poorly understood and difficult to control. In this review, we will first discuss experimental studies that have elucidated roles for parameters such as nanoparticle size and shape, targeting ligand and target receptor densities, and monovalent binding kinetics on multivalent nanoparticle adhesion efficiency and cellular internalization. Although such experimental studies are very insightful, information is limited and confounded by numerous differences across experimental systems. Thus, we focus the second part of the review on theoretical aspects of binding, including kinetics, biomechanics, and transport physics. Finally, we discuss various computational and simulation studies of nanoparticle adhesion, including advanced treatments that compare directly to experimental results. Future work will ideally continue to combine experimental data and advanced computational studies to extend our knowledge of multivalent adhesion, as well as design the most powerful nanoparticle-based agents to treat cancer.

Maximizing the speed and efficiency at which single cells can be liberated from tissues would dramatically advance cell-based diagnostics and therapies. Conventional methods involve numerous manual processing steps and long enzymatic digestion times, yet are still inefficient. In previous work, we developed a microfluidic device with a network of branching channels to improve the dissociation of cell aggregates into single cells. However, this device was not tested on tissue specimens, and further development was limited by high cost and low feature resolution. In this work, we utilized a single layer, laser micro-machined polyimide film as a rapid prototyping tool to optimize the design of our microfluidic channels to maximize dissociation efficiency. This resulted in a new design with smaller dimensions and a shark fin geometry, which increased recovery of single cells from cancer cell aggregates. We then tested device performance on mouse kidney tissue, and found that optimal results were obtained using two microfluidic devices in series, the larger original design followed by the new shark fin design as a final polishing step. We envision our microfluidic dissociation devices being used in research and clinical settings to generate single cells from various tissue specimens for diagnostic and therapeutic applications.