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Accurate dispensing system for single oocytes using air ejection.

ABSTRACT: In this study, we propose a new approach to increase the success rate of single-oocyte dispensing and investigate the subsequent viability of the dispensed oocytes. We used a pair of capacitance sensors placed in a microfluidic chip to detect the oocyte, and custom-designed a special buffer zone in the microchannel to decelerate the flow velocity and reduce the hydraulic pressure acting on the oocyte. In the buffer zone, a semicircular bay, formed by equally spaced micro-pillars, is used to stop the oocyte at the dispensing nozzle hole. Finally, the oocyte is ejected by airflow to the culture array. The novel feature of the developed microfluidic system is that the extraordinary improvement in success rate is accompanied by a lack of change in oocyte survival rate (as assessed by a comparison of survival rates before and after the dispensing procedure). By using this device, we achieved a highly accurate single-oocyte dispensing process with a success rate of 100%. The oocyte survival rate is approximately 70%, regardless of whether or not the oocyte is dispensed. The newly proposed system has the advantages of high operation speed and potential usage for two-dimensional micropatterning.

SUBMITTER: Feng L

PROVIDER: S-EPMC3799720 | biostudies-other | 2013

REPOSITORIES: biostudies-other

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Project description:As of today, the majority of environmental microorganisms remain uncultured. They are therefore referred to as "microbial dark matter." In the recent past, cultivation-independent methods like single-cell genomics (SCG) enabled the discovery of many previously unknown microorganisms, among them the Patescibacteria/Candidate Phyla Radiation (CPR). This approach was shown to be complementary to metagenomics, however, the development of additional and refined sorting techniques beyond the most commonly used fluorescence-activated cell sorting (FACS) is still desirable to enable additional downstream applications. Adding image information on the number and morphology of sorted cells would be beneficial, as would be minimizing cell stress caused by sorting conditions such as staining or pressure. Recently, a novel cell sorting technique has been developed, a microfluidic single-cell dispenser, which assesses the number and morphology of the cell in each droplet by automated light microscopic processing. Here, we report for the first time the successful application of the newly developed single-cell dispensing system for label-free isolation of individual bacteria from a complex sample retrieved from a wastewater treatment plant, demonstrating the potential of this technique for single cell genomics and other alternative downstream applications. Genome recovery success rated above 80% with this technique-out of 880 sorted cells 717 were successfully amplified. For 50.1% of these, analysis of the 16S rRNA gene was feasible and led to the sequencing of 50 sorted cells identified as Patescibacteria/CPR members. Subsequentially, 27 single amplified genomes (SAGs) of 15 novel and distinct Patescibacteria/CPR members, representing yet unseen species, genera and families could be captured and reconstructed. This phylogenetic distinctness of the recovered SAGs from available metagenome-assembled genomes (MAGs) is accompanied by the finding that these lineages-in whole or in part-have not been accessed by genome-resolved metagenomics of the same sample, thereby emphasizing the importance and opportunities of SCGs.

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Accurate dispensing system for single oocytes using air ejection.

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