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

Gene-targeted microfluidic cultivation validated by isolation of a gut bacterium listed in Human Microbiome Project's Most Wanted taxa

This paper describes a microfluidics-based workflow for genetically targeted isolation and cultivation of microorganisms from complex clinical samples. Data sets from high-throughput sequencing suggest the existence of previously unidentified bacterial taxa and functional genes with high biomedical importance. Obtaining isolates of these targets, preferably in pure cultures, is crucial for advancing understanding of microbial genetics and physiology and enabling physical access to microbes for further applications. However, the majority of microbes have not been cultured, due in part to the difficulties of both identifying proper growth conditions and characterizing and isolating each species. We describe a method that enables genetically targeted cultivation of microorganisms through a combination of microfluidics and on- and off-chip assays. This method involves (i) identification of cultivation conditions for microbes using growth substrates available only in small quantities as well as the correction of sampling bias using a “chip wash” technique; and (ii) performing on-chip genetic assays while also preserving live bacterial cells for subsequent scale-up cultivation of desired microbes, by applying recently developed technology to create arrays of individually addressable replica microbial cultures. We validated this targeted approach by cultivating a bacterium, here referred to as isolate microfluidicus 1, from a human cecal biopsy. Isolate microfluidicus 1 is, to our knowledge, the first successful example of targeted cultivation of a microorganism from the high-priority group of the Human Microbiome Project’s “Most Wanted” list, and, to our knowledge, the first cultured representative of a previously unidentified genus of the Ruminococcaceae family