Femtoliter droplet confinement of Streptococcus pneumoniae: bacterial genetic transformation by cell-cell interaction in droplets.

Streptococcus pneumoniae (pneumococcus), a deadly bacterial human pathogen, uses genetic transformation to gain antibiotic resistance. Genetic transformation begins when a pneumococcal strain in a transient specialized physiological state called competence, attacks and lyses another strain, releasing DNA, taking up fragments of the liberated DNA, and integrating divergent genes into its genome. While many steps of the process are known and generally understood, the precise mechanism of this natural genetic transformation is not fully understood and the current standard strategies to study it have limitations in specifically controlling and observing the process in detail. To overcome these limitations, we have developed a droplet microfluidic system for isolating individual episodes of bacterial transformation between two confined cells of pneumococcus. By encapsulating the cells in a 10 µm diameter aqueous droplet, we provide an improved experimental model of genetic transformation, as both participating cells can be identified, and the released DNA is spatially restricted near the attacking strain. Specifically, the bacterial cells, one rifampicin (R) resistant, the other novobiocin (N) and spectinomycin (S) resistant were encapsulated in droplets carried by the fluorinated oil FC-40 with 5% surfactant and allowed to carry out competence-specific attack and DNA uptake (and consequently gain antibiotic resistances) within the droplets. The droplets were then broken, and recombinants were recovered by selective plating with antibiotics. The new droplet system encapsulated 2 or more cells in a droplet with a probability up to 71%, supporting gene transfer rates comparable to standard mixtures of unconfined cells. Thus, confinement in droplets allows characterization of natural genetic transformation during a strictly defined interaction between two confined cells.

Open Design 3D-Printable Adjustable Micropipette that Meets the ISO Standard for Accuracy.

Scientific communities are drawn to the open source model as an increasingly utilitarian method to produce and share work. Initially used as a means to develop freely-available software, open source projects have been applied to hardware including scientific tools. Increasing convenience of 3D printing has fueled the proliferation of open labware projects aiming to develop and share designs for scientific tools that can be produced in-house as inexpensive alternatives to commercial products. We present our design of a micropipette that is assembled from 3D-printable parts and some hardware that works by actuating a disposable syringe to a user-adjustable limit. Graduations on the syringe are used to accurately adjust the set point to the desired volume. Our open design printed micropipette is assessed in comparison with a commercial pipette and meets the ISO 8655 standards.

A 3D-Printed Oxygen Control Insert for a 24-Well Plate.

3D printing has emerged as a method for directly printing complete microfluidic devices, although printing materials have been limited to oxygen-impermeable materials. We demonstrate the addition of gas permeable PDMS (Polydimethylsiloxane) membranes to 3D-printed microfluidic devices as a means to enable oxygen control cell culture studies. The incorporation of a 3D-printed device and gas-permeable membranes was demonstrated on a 24-well oxygen control device for standard multiwell plates. The direct printing allows integrated distribution channels and device geometries not possible with traditional planar lithography. With this device, four different oxygen conditions were able to be controlled, and six wells were maintained under each oxygen condition. We demonstrate enhanced transcription of the gene VEGFA (vascular endothelial growth factor A) with decreasing oxygen levels in human lung adenocarcinoma cells. This is the first 3D-printed device incorporating gas permeable membranes to facilitate oxygen control in cell culture.

Oxygen control with microfluidics.

Cellular function and behavior are affected by the partial pressure of O2, or oxygen tension, in the microenvironment. The level of oxygenation is important, as it is a balance of oxygen availability and oxygen consumption that is necessary to maintain normoxia. Changes in oxygen tension, from above physiological oxygen tension (hyperoxia) to below physiological levels (hypoxia) or even complete absence of oxygen (anoxia), trigger potent biological responses. For instance, hypoxia has been shown to support the maintenance and promote proliferation of regenerative stem and progenitor cells. Paradoxically, hypoxia also contributes to the development of pathological conditions including systemic inflammatory response, tumorigenesis, and cardiovascular disease, such as ischemic heart disease and pulmonary hypertension. Current methods to study cellular behavior in low levels of oxygen tension include hypoxia workstations and hypoxia chambers. These culture systems do not provide oxygen gradients that are found in vivo or precise control at the microscale. Microfluidic platforms have been developed to overcome the inherent limits of these current methods, including lack of spatial control, slow equilibration, and unachievable or difficult coupling to live-cell microscopy. The various applications made possible by microfluidic systems are the topic of this review. In order to understand how the microscale can be leveraged for oxygen control of cells and tissues within microfluidic systems, some background understanding of diffusion, solubility, and transport at the microscale will be presented in addition to a discussion on the methods for measuring the oxygen tension in microfluidic channels. Finally the various methods for oxygen control within microfluidic platforms will be discussed including devices that rely on diffusion from liquid or gas, utilizing on-or-off-chip mixers, leveraging cellular oxygen uptake to deplete the oxygen, relying on chemical reactions in channels to generate oxygen gradients in a device, and electrolytic reactions to produce oxygen directly on chip.