Individually addressable arrays of replica microbial cultures enabled by splitting SlipChips

Isolating microbes carrying genes of interest from environmental samples is important for applications in biology and medicine. However, this involves the use of genetic assays that often require lysis of microbial cells, which is not compatible with the goal of obtaining live cells for isolation and culture. This paper describes the design, fabrication, biological validation, and underlying physics of a microfluidic SlipChip device that addresses this challenge. The device is composed of two conjoined plates containing 1000 microcompartments, each comprising two juxtaposed wells, one on each opposing plate. Single microbial cells are stochastically confined and subsequently cultured within the microcompartments. Then, we split each microcompartment into two replica droplets, both containing microbial culture, and then controllably separate the two plates while retaining each droplet within each well. We experimentally describe the droplet retention as a function of capillary pressure, viscous pressure, and viscosity of the aqueous phase. Within each pair of replicas, one can be used for genetic analysis, and the other preserves live cells for growth. This microfluidic approach provides a facile way to cultivate anaerobes from complex communities. We validate this method by targeting, isolating, and culturing Bacteroides vulgatus, a core gut anaerobe, from a clinical sample. To date, this methodology has enabled isolation of a novel microbial taxon, representing a new genus. This approach could also be extended to the study of other microorganisms and even mammalian systems, and may enable targeted retrieval of solutions in applications including digital PCR, sequencing, single cell analysis, and protein crystallization

Drug Resistance in Eukaryotic Microorganisms

Eukaryotic microbial pathogens are major contributors to illness and death globally. Although much of their impact can be controlled by drug therapy as with prokaryotic microorganisms, the emergence of drug resistance has threatened these treatment efforts. Here, we discuss the challenges posed by eukaryotic microbial pathogens and how these are similar to, or differ from, the challenges of prokaryotic antibiotic resistance. The therapies used for several major eukaryotic microorganisms are then detailed, and the mechanisms that they have evolved to overcome these therapies are described. The rapid emergence of resistance and the restricted pipeline of new drug therapies pose considerable risks to global health and are particularly acute in the developing world. Nonetheless, we detail how the integration of new technology, biological understanding, epidemiology and evolutionary analysis can help sustain existing therapies, anticipate the emergence of resistance or optimize the deployment of new therapies

Targeting the trypanosome kinetochore with CLK1 protein kinase inhibitors

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Digital biology and chemistry

This account examines developments in "digital" biology and chemistry within the context of microfluidics, from a personal perspective. Using microfluidics as a frame of reference, we identify two areas of research within digital biology and chemistry that are of special interest: (i) the study of systems that switch between discrete states in response to changes in chemical concentration of signals, and (ii) the study of single biological entities such as molecules or cells. In particular, microfluidics accelerates analysis of switching systems (i.e., those that exhibit a sharp change in output over a narrow range of input) by enabling monitoring of multiple reactions in parallel over a range of concentrations of signals. Conversely, such switching systems can be used to create new kinds of microfluidic detection systems that provide "analog-to-digital" signal conversion and logic. Microfluidic compartmentalization technologies for studying and isolating single entities can be used to reconstruct and understand cellular processes, study interactions between single biological entities, and examine the intrinsic heterogeneity of populations of molecules, cells, or organisms. Furthermore, compartmentalization of single cells or molecules in "digital" microfluidic experiments can induce switching in a range of reaction systems to enable sensitive detection of cells or biomolecules, such as with digital ELISA or digital PCR. This "digitizing" offers advantages in terms of robustness, assay design, and simplicity because quantitative information can be obtained with qualitative measurements. While digital formats have been shown to improve the robustness of existing chemistries, we anticipate that in the future they will enable new chemistries to be used for quantitative measurements, and that digital biology and chemistry will continue to provide further opportunities for measuring biomolecules, understanding natural systems more deeply, and advancing molecular and cellular analysis. Microfluidics will impact digital biology and chemistry and will also benefit from them if it becomes massively distributed

The Modular μSiM: a Mass Produced, Rapidly Assembled, and Reconfigurable Platform for the Study of Barrier Tissue Models In Vitro.

Advanced in vitro tissue chip models can reduce and replace animal experimentation and may eventually support "on-chip" clinical trials. To realize this potential, however, tissue chip platforms must be both mass-produced and reconfigurable to allow for customized design. To address these unmet needs, we introduce an extension of our μSiM (microdevice featuring a silicon-nitride membrane) platform. The modular μSiM (m-μSiM) uses mass-produced components to enable rapid assembly and reconfiguration by laboratories without knowledge of microfabrication. We demonstrate the utility of the m-μSiM by establishing an hiPSC-derived blood-brain barrier (BBB) in bioengineering and non-engineering, brain barriers focused laboratories. We develop and validate in situ and sampling-based assays of small molecule diffusion as a measure of barrier function. BBB properties show excellent interlaboratory agreement and match expectations from literature, validating the m-μSiM as a platform for barrier models and demonstrating successful dissemination of components and protocols. We then demonstrate the ability to quickly reconfigure the m-μSiM for co-culture and immune cell transmigration studies through addition of accessories and/or quick exchange of components. Because the development of modified components and accessories is easily achieved, custom designs of the m-μSiM should be accessible to any laboratory desiring a barrier-style tissue chip platform. This article is protected by copyright. All rights reserved