A programmable microenvironment for cellular studies via microfluidics-generated double emulsions

Abstract

High throughput cellular studies require small sample volume to reduce costs and enhance sensitivity. Microfluidics-generated water-in-oil (W/O) single emulsion droplet systems, in particular, provide uniform, well defined and discrete microenvironment for cell culture, screening, and sorting. However, these single emulsion droplets are incapable of continuous supply of nutrient molecule and are not compatible with aqueous phase-based analysis. A solution is to entrap W/O droplets in another aqueous phase, forming water-in-oil-in-water (W/O/W) double emulsions. The external aqueous phase efficiently prevents desiccation and reduces the amount of organic component, and yet retaining the advantages of compartmentalization. The internal environment can also be programmed dynamically without the need of rupturing the droplets. In this study, we explore the potential application of W/O/W double emulsion droplets for cell cultivation, genetic activation and study of more complicated biological events such as bacteria quorum-sensing as an example. This study demonstrates the advantages and potential application of double emulsion for the study of complex biological processes.

Figure 1

Chip design and double emulsion generation. A) A schematic of a two-chip setup for double emulsion formation: W/O droplets were firstly formed in an untreated PDMS channel (Chip 1) and then fed to a hydrophilic treated channel (Chip 2) to generate W/O/W double emulsion droplets; B) Number of W/O droplets encapsulated into double emulsion was modulated by the flow rate of continuous phase (water). (Scale bar: 100 μm)

Figure 2

Selective chemical transport across the oil phase of double emulsion. Diffusion of rhodamine B (A) and rhodamine B labeled BSA (B) from the core of double emulsion measured by change of fluorescence intensity per droplet. r is the distance from the center of emulsion.

Figure 3

aTC diffusion and activation of GFP expression. A) Schematic illustration of the experiment design. GFP was expressed under aTC dependent promoter. Bacteria carry this expressing vector was encapsulated in the core without aTC. aTc was then added to the external medium, which diffuse into core of droplets to activate GFP expression; B) Fluorescence microscope images of droplets containing bacteria without aTC addition (top panel) and with aTC addition (bottom panel). Images were taken 1 hour after cell encapsulation. (Scale bar: 100 μm)

Figure 4

aTC dependent GFP activation. A) Fluorescence microscope image showing GFP expression overtime after aTC addition; B) Relative GFP level per droplet after aTC addition as a function of time; C) Relative GFP level per droplet as a function of aTC concentration (Data were taken 2 hours after aTC addition). (Scale bar: 100 μm)

Figure 5

Bacteria growth monitored by flow cytometry. Constitutive GFP expressing bacteria was diluted to PBS solution, and encapsulated in double emulsion droplets. These droplets were then suspended to PBS solution (left) or medium solution (right) to compare bacteria growth over time.

Figure 6

Quorum-sensing activity in double emulsion microenvironment. A) GFP fluorescence intensity per droplet over time. B) Fluorescence microscope images showing change of GFP-expressing bacteria population over time. GFP-expressing bacteria were co-cultured with bacteria-expressing quorum sensing circuit. When the bacteria population reached a threshold density around 5 hours, the quorum-sensing bacteria released lytic protein to kill the bacteria in the droplets. The depletion caused the reduction of lytic protein expression and recovery of bacteria growth. (Scale bar: 50 μm)