(Nanowerk Spotlight) Scientists manipulating living cells face a fundamental challenge: working with thousands of samples at the nanoliter scale – volumes measured in billionths of a liter. At this scale, researchers can dramatically reduce the volume of expensive reagents and biological materials needed while increasing the number of experiments they can run simultaneously. However, precisely controlling such small volumes while maintaining living cells has proven technologically difficult.
Standard laboratory techniques use plastic plates containing small wells, each typically holding 100 to 200 microliters of cell-growing liquid. While effective for basic experiments, these relatively large volumes become impractical when scaling up to thousands of samples. A typical screening experiment using traditional well plates might require milliliters of costly reagents and cellular materials. Even advanced robotic systems must process these wells sequentially, making large-scale experiments time-consuming and expensive.
Droplet microarrays (DMAs) offer a solution through extreme miniaturization. These specialized glass slides feature precisely patterned surfaces that hold thousands of individual 300-nanoliter droplets in specific locations without physical walls. This represents a 100- to 1000-fold reduction in volume compared to traditional well plates. The DMA’s open design allows easier access to samples than enclosed wells, but this miniaturization creates new technical challenges. Changing nutrient solutions or moving cells between such small droplets without cross-contamination has limited experiments to just three days before cells begin dying.
DMA and sandwiching strategy. A.1) Image of the DMA submerged in water showing the formation of nanoliter droplets due to its hydrophilic spots on a superhydrophobic background. A.2) DMA dimensions match those of a standard microscope slide. A.3) Microscopy image featuring 6 spots: showcasing superhydrophobic borders and hydrophilic spots within the DMA. A.4) Microscopy images capturing diverse cell culture models on DMA substrate. B) Schematic depicting the assembly of the sandwich-like structure using a DMA. The procedure is illustrated from left to right. (Before sandwiching) The initial inset shows a side view of the DMA containing 300 nL droplets, alongside an illustration showing how the droplets are positioned. This slide is hereafter referred to as the donor slide. (Sandwiching) The insets show the DMA sandwiched by a counter slide, which is referred to as the acceptor slide. When positioned 0.4 mm above the base of the droplets using an adapter, the acceptor slide contacts the droplets, forming a capillary bridge. As the acceptor slide is removed, the capillary bridge elongates until instability occurs at its neck, resulting in two isolated droplets. (After sandwiching) Side view image of the DMA post-sandwiching, showing a reduction in droplet volume. B.1.2.3) Schematic representation of the different sandwich-based applications developed in this paper. Volume removal allows to remove a portion of the droplet volume using a homogeneous surface as an acceptor slide. High-throughput Sampling (HT Sampling) employs a DMA slide as an acceptor slide, with identical dimensions to the donor slide, for liquid removal, thereby producing a copy of the donor slide. For the spheroids transfer DMA slides were used as a donor and acceptor slides to transfer spheroids between them using gravity. (Image: Reprinted with permission by Wiley-VCH Verlag)
The method employs two glass slides positioned using a custom 3D-printed frame. The bottom slide contains the cell-holding nanoliter droplets, while the top slide extracts or transfers liquid through controlled splitting of these droplets. When brought together, each droplet forms a temporary bridge between the slides. As they separate, surface tension forces cause the droplets to split predictably, allowing researchers to remove spent medium and add fresh nutrients without disturbing the cells.
This approach extends cell survival times significantly. The team maintained both adherent cells (which attach to surfaces) and suspension cells (which float freely) alive for seven days through regular nutrient replacement. More impressively, they kept spheroids – ball-shaped cell clusters that better mimic natural tissues – healthy for 14 days, nearly five times longer than previous methods.
The system’s precision allows researchers to extract samples from thousands of droplets simultaneously. The team demonstrated this by measuring lactate dehydrogenase, an enzyme released by dying cells, across hundreds of samples in seconds – a process that would take hours with traditional methods.
The technique also enables controlled movement of spheroids between different slides with 95% success rate. Researchers can combine multiple spheroids into larger structures, a crucial capability for tissue engineering. The method proves particularly valuable for suspension cells and spheroids, which typically resist manipulation without damage. Video analysis shows cells remaining undisturbed at the droplet base during liquid exchange.
The researchers addressed practical challenges including evaporation and contamination. Their system maintains sterile conditions and prevents droplet drying during long-term experiments. The custom 3D-printed frame ensures precise alignment while allowing direct microscope observation.
This advancement significantly expands DMA applications in drug screening and tissue engineering. The platform can maintain different cell types in precise arrangements, essential for creating complex tissue structures that better represent human biology. Its combination of nanoliter-scale operation, high throughput, and precise control provides new capabilities for studying cell behavior and developing therapeutic applications.
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