Devices for Biological Systems: On-Chip Horizontal Gene Transfer and 3D-Printed Microfluidic Applications
thesisposted on 2017-11-01, 00:00 authored by Martin D. Brennan
This thesis covers the development of three devices for investigating biological systems: A 3D-printed oxygen control insert for a 24-well plate, an open source 3D-printable adjustable micropipette, and a droplet encapsulation device to isolate and investigate genetic transfer between pairs of S. pneumoniae. Microfluidic platforms have been developed to provide more physiologically relevant oxygen environments for cell studies. Typically, building these platforms involves manual fabrication of microfluidic chips, a process that is time consuming, has a high failure rate and only produces limited geometries. Recently 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 a 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 that can be functionalized to control oxygen in cell culture. 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, the open source model has 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 cheap 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 to a commercial pipette and meets ISO 8655 standards for accuracy and precision. In genetic transformation (GT), cells take up and incorporate DNA from the environment. GT allows rapid alterations of the genome resulting in persistent infections as polyclonal S. pneumoniae biofilms share genes to overcome immune response and transfer antibiotic resistance. In vitro, GT is studied in suspensions of billions of cells and can be used to assay transfer of traits between strains, such as antibiotic resistance and virulence, but does not reproduce the rapid and extensive of transfer observed in vivo. A flow focusing droplet generating device is demonstrated to encapsulate S. pneumoniae in droplets of M9 minimal medium. The addition of a coverslip layer to the microfluidic incubation chamber prevents evaporation of droplets which allows imaging of encapsulated cells on-chip that self organize into a monolayer. Imaging of cells in droplets confirms an occupancy distribution average of 2.5 cells per droplet and a mode of 2 cells per droplet.