Microbiome culture in droplet using dsurf surfactant
Biomillenia’s microbiome-on-a-chip technology uses droplet generation to create bacterial cell banks, evaluate microorganisms for desired phenotypes and to find new routes to prevent and treat dysbiosis. Most of the microfluidic droplet techniques require biocompatible surfactants to keep the droplets stable but it can be affected by growth of bacteria in droplets. Hence, choosing the optimal surfactant for long-term incubation while being biocompatible for a wide spectrum of bacterial species is essential.
Here, the performance of three commonly used surfactants are compared at three different concentrations. The droplet stability over time and the droplet occupation rate is determined by encapsulating a microbial community derived from human skin.
This application note was made in collaboration with Biomillenia. Biomillenia is a team of experts in microfluidics, microbiology, molecular biology and bioinformatics who are passionate about harnessing smart microbes to restore the body’s healthy balance and help prevent lifestyle diseases.
Materials and methods
Novec HFE7500 (3M) containing 5%, 2% or 0.5% (w/w) concentration of Competitor 1 surfactant, Competitor 2 surfactant, or dSurf (Fluigent), respectively.
A microbial sample collected from skin of healthy volunteers was used for droplet generation. A general microbial skin sample, as opposed to single strain like E. coli, was used here because a bacterial community consisting of many species better represents the experimental condition for microbiological research. The microbial skin sample was prepared according to Biomillenia’s proprietary standard sample preparation workflow and was suspended in a standard medium for skin microbes containing 0.5% (v/v) TWEEN80. TWEEN80 is a lipophilic molecule in the aqueous phase that interfes with the droplet stabilizing characteristics of the surfactants.
The droplets were prepared on Biomillenia’s proprietary microfluidic platform with PDMS chips. The two liquid phases were controlled during droplet generation by the use of high precision pressure pumps (Fluigent, Flow EZ). Droplets were generated at frequencies of 7-10 kHz. The droplet volume was set to 20 pL. Droplets were collected and incubated in custom made vessels at 37 °C allowing for the storage of the droplets without exposing to a direct gas interface.
From the droplet collection vessel, a small number of droplets were sampled to check for droplet stability and bacterial occupation at 0, 1, 3 and 7 days of incubation. For imaging by microscopy, droplets were spread onto a monolayer surrounded by the respective oil-surfactant combination.
Droplet stability and bacteria occupancy
The collected droplets were spread in Biomillenia’s proprietary observation chip for best imaging of microbial occupation in droplets after 7 days incubation at 37 °C. As shown in the figure below, a monolayer of droplets surrounded by the respective oil-surfactant combination was imaged microscopically.
Surfactant concentrations of 2% and higher result in stable populations over 7 days at 37 °C for dSURF and Competitor 2 while for Competitor 1 a concentration of 5% is required to reach similar results
The stability of microfluidic emulsions strongly depends on the content of droplets and its interplay with the surfactant used. Hydrophobic compounds in the aqueous phase, along with microbial growth of various species present far from ideal conditions for microfluidic droplets, but exemplify well the experimental challenges encountered when microfluidic techniques are truly applied in microbiology. Hence, surfactants are needed that help to accommodate those complex sample characteristics.
In conclusion, dSURF surfactant for droplet microfluidics performs very well compared to widely established microfluidic surfactants and is highly suitable for complex biological applications
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