Background: The unusual nuclear shape of neutrophils has been speculated to facilitate their passage through confined spaces. Results: Levels of nuclear protein lamin A modulate cell passage through micron-scale pores. Conclusion:The unique protein composition of neutrophil nuclei facilitates their deformation; lobulated nuclear shape is not essential. Significance: Altered nuclear envelope composition, as reported in cancer cells, could impact cell passage through physiological gaps.

Digital microfluidic (DMF) liquid handling includes active (electrostatic) and passive (surface tension) mechanisms for reagent dispensing. Here we implement a simple and straightforward Teflon-AF liftoff protocol for patterning hydrophilic sites on a two-plate device for precise passive dispensing of reagents forming virtual microwells--an analogy to the wells found on a microtitre plate. We demonstrate here that devices formed using these methods are capable of reproducible dispensing of volumes ranging from ~80 to ~800 nL, with CVs of 0.7% to 13.8% CV. We demonstrate that passive dispensing is compatible with DMF operation in both air and oil, and provides for improved control of dispensed nano- and micro- litre volumes when compared to active electrostatic dispensing. Further, the technique is advantageous for cell culture and we report the first example of reagent dispensing on a single-plate DMF device. We anticipate this method will be useful for a wide range of applications--particularly those involving adherent cell culture and analysis.

Three-dimensional (3D) hydrogel structures are finding use in fundamental studies of self-assembly, rheology, and 3D cell culture. Most techniques for 3D hydrogel formation are 'single pot', in which gels are not addressable after formation. For many applications, it would be useful to be able to form arrays of gels bearing mixtures of constituents and/or formed from composites of different gel materials. Here, in response to this challenge, we introduce a digital microfluidic method for 'on-demand' formation of arrays of microgels bearing arbitrary contents and shapes. On formation of the gels, each microgel is individually addressable for reagent delivery and analysis. We demonstrate the utility of the method for 3D cell culture and higher-order tissue formation by implementing the first sub-microlitre recapitulation of 3D kidney epithelialization. We anticipate this platform will enable new research that can exploit the flexible nature of this technique for forming and addressing arrays of hydrogels with unique geometries and contents.

Digital microfluidics (DMF) is a technology that facilitates electrostatic manipulation of discrete nano- and micro-litre droplets across an array of electrodes, which provides the advantages of single sample addressability, automation, and parallelization. There has been considerable interest in recent years in using DMF for cell culture and analysis, but previous studies have used immortalized cell lines. We report here the first digital microfluidic method for primary cell culture and analysis. A new mode of "upside-down" cell culture was implemented by patterning the top plate of a device using a fluorocarbon liftoff technique. This method was useful for culturing three different primary cell types for up to one week, as well as implementing a fixation, permeabilization, and staining procedure for F-actin and nuclei. A multistep assay for monocyte adhesion to endothelial cells (ECs) was performed to evaluate functionality in DMF-cultured primary cells and to demonstrate co-culture using a DMF platform. Monocytes were observed to adhere in significantly greater numbers to ECs exposed to tumor necrosis factor (TNF)-α than those that were not, confirming that ECs cultured in this format maintain in vivo-like properties. The ability to manipulate, maintain, and assay primary cells demonstrates a useful application for DMF in studies involving precious samples of cells from small animals or human patients.

The protection of groundwater supplies from microbial contamination necessitates a solid understanding of the key factors controlling the migration and retention of pathogenic organisms through the subsurface environment. The transport behavior of five waterborne pathogens was examined using laboratory-scale columns packed with clean quartz at two solution ionic strengths (10 mM and 30 mM). Escherichia coli O157:H7 and Yersinia enterocolitica were selected as representative Gram-negative pathogens, Enterococcus faecalis was selected as a representative Gram-positive organism, and two cyanobacteria (Microcystis aeruginosa and Anabaena flos-aquae) were also studied. The five organisms exhibit differing attachment efficiencies to the quartz sand. The surface (zeta) potential of the microorganisms was characterized over a broad range of pH values (2-8) at two ionic strengths (10 mM and 30 mM). These measurements are used to evaluate the observed attachment behavior within the context of the DLVO theory of colloidal stability. To better understand the possible link between bacterial transport in model quartz sand systems and natural soil matrices, additional experiments were conducted with two of the selected organisms using columns packed with loamy sand obtained from an agricultural field. This investigation highlights the need for further characterization of waterborne pathogen surface properties and transport behavior over a broader range of environmentally relevant conditions.