Two-Layer Droplet Arrays Enable Dynamic Manipulation of Cell Microenvironment During High-Throughput Bacterial...

Area of Science:

• Biotechnology
• Microfluidics
• Cell Biology

Background:

• Droplet-based microfluidics enables high-throughput chemical and biological analyses.
• Controlling the microenvironment of droplets is crucial for dynamic biological cultivation.

Purpose of the Study:

• To present a two-layer droplet platform for flexible microenvironment manipulation.
• To enable dynamic biological cultivation and high-throughput screening in nanoliter droplets.

Main Methods:

• Generation of agarose droplets (≈2.0 nL) encapsulating bacterial cells.
• Deposition of aqueous droplets (≈3.7 nL) on top to form a two-layer system.
• Fast chemical exchange (≈15 s) and substance extraction via buffer exchange.

Main Results:

• Demonstrated rapid chemical exchange and efficient extraction of substances from droplets.
• Enabled antibiotic drug testing under static and transient exposure, revealing single-cell heterogeneity.
• Facilitated long-term cultivation (72 h) of slow-growing microorganisms through medium replenishment.

Conclusions:

• The two-layer droplet platform offers precise control over the microenvironment for biological assays.
• This technology supports high-throughput screening, dynamic exposure studies, and long-term cell cultivation.
• The platform reveals single-cell heterogeneity in bacterial responses to antibiotics.