Limitations imposed by conventional analytical technologies for cell biology, such as flow cytometry or microplate imaging, are often prohibitive for the kinetic analysis of single-cell responses to therapeutic compounds. In this paper, we describe the application of a microfluidic array to the real-time screening of anti-cancer drugs against arrays of single cells. The microfluidic platform comprises an array of micromechanical traps, designed to passively corral individual non-adherent cells. This platform, fabricated in the biologically compatible elastomer poly(dimethylsiloxane), PDMS, enables hydrodynamic trapping of cells in low shear stress zones, enabling time-lapse studies of non-adherent hematopoietic cells. Results indicate that these live-cell, microfluidic microarrays can be readily applied to kinetic analysis of investigational anti-cancer agents in hematopoietic cancer cells, providing new opportunities for automated microarray cytometry and higher-throughput screening. We also demonstrate the ability to quantify on-chip the anti-cancer drug induced apoptosis. Specifically, we show that with small numbers of trapped cells (~300) under careful serial observation we can achieve results with only slightly greater statistical spread than can be obtained with single-pass flow cytometer measurements of 15,000 – 30,000 cells.

In addition to the annular or boundary lipids that surround the transmembrane surface of the potassium channel KcsA from Streptomyces lividans, x-ray crystallographic studies have detected one anionic lipid molecule bound at each protein-protein interface in the homotetrameric structure, at sites referred to as nonannular sites. The binding constant for phosphatidylglycerol at the nonannular sites has been determined using fluorescence quenching methods with a mutant of KcsA lacking the normal three lipid-exposed Trp residues. Binding is weak, with a binding constant of 0.42 +/- 0.06 in units of mol fraction, implying that the nonannular sites will only be approximately 70% occupied in bilayers of 100% phosphatidylglycerol. However, the nonannular sites show high selectivity for anionic lipids over zwitterionic lipids, and it is suggested that a change in packing at the protein-protein interface leads to a closing of the nonannular binding site in the unbound state. Increasing the anionic lipid content of the membrane leads to a large increase in open channel probability, from approximately 2.5% in the presence of 25 mol % phosphatidylglycerol to approximately 62% in 100 mol % phosphatidylglycerol. The relationship between open channel probability and phosphatidylglycerol content shows cooperativity. The data are consistent with a model in which three or four of the four nonannular sites in the KcsA homotetramer have to be occupied by anionic lipid for the channel to open. The conductance of the open channel increases with increasing concentration of anionic lipid, an effect possibly due to effects of anionic lipid on the concentration of K(+) close to the membrane surface.

The alternative splicing of the tau gene, MAPT, generates six protein isoforms in the adult human central nervous system (CNS). Tau splicing is developmentally regulated and dysregulated in disease. Mutations in MAPT that alter tau splicing cause frontotemporal dementia (FTD) with tau pathology, providing evidence for a causal link between altered tau splicing and disease. The use of induced pluripotent stem cell (iPSC)-derived neurons has revolutionized the way we model neurological disease in vitro. However, as most tau mutations are located within or around the alternatively spliced exon 10, it is important that iPSC–neurons splice tau appropriately in order to be used as disease models. To address this issue, we analyzed the expression and splicing of tau in iPSC-derived cortical neurons from control patients and FTD patients with the 10 + 16 intronic mutation in MAPT. We show that control neurons only express the fetal tau isoform (0N3R), even at extended time points of 100 days in vitro. Neurons from FTD patients with the 10 + 16 mutation in MAPT express both 0N3R and 0N4R tau isoforms, demonstrating that this mutation overrides the developmental regulation of exon 10 inclusion in our in vitro model. Further, at extended time points of 365 days in vitro, we observe a switch in tau splicing to include six tau isoforms as seen in the adult human CNS. Our results demonstrate the importance of neuronal maturity for use in in vitro modeling and provide a system that will be important for understanding the functional consequences of altered tau splicing.

Artificially reproducing cellular environments is a key aim of synthetic biology, which has the potential to greatly enhance our understanding of cellular mechanisms. Microfluidic and lab-on-a-chip (LOC) techniques, which enable the controlled handling of sub-microlitre volumes of fluids in an automated and high-throughput manner, can play a major role in achieving this by offering alternative and powerful methodologies in an on-chip format. Such techniques have been successfully employed over the last twenty years to provide innovative solutions for chemical analysis and cell-, molecular- and synthetic- biology. In the context of the latter, the formation of artificial cell membranes (or artificial lipid bilayers) that incorporate membrane proteins within miniaturised LOC architectures offers huge potential for the development of highly sensitive molecular sensors and drug screening applications. The aim of this review is to give a comprehensive and critical overview of the field of microsystems for creating and exploiting artificial lipid bilayers. Advantages and limitations of three of the most popular approaches, namely suspended, supported and droplet-based lipid bilayers, are discussed. Examples are reported that show how artificial cell membrane microsystems, by combining together biological procedures and engineering techniques, can provide novel methodologies for basic biological and biophysical research and for the development of biotechnology tools.