PARTICLE-TEMPLATED EMULSIFICATION IN WELL PLATES
Researchers at UCSF have developed a new method for emulsifying droplet libraries that is highly scalable.
In the past decade, droplet microfluidic technology has revolutionized the sequencing space via single cell RNA-sequencing (scRNA-seq). This technology has vastly increased the resolution of RNA expression in heterogenous tissues and enabled the identification of important molecular signatures and new cell types. Despite their significant contributions to the field, droplet microfluidics have several major limitations that make their implementation in a larger scale difficult. Combinatorial applications for generating droplet libraries often require each sample to be emulsified in a separate fluidic device to ensure low risk of cross-contamination, which is costly and labor intensive. Some approaches allow for the emulsification of samples in separate devices that then interface with well plates, increasing scalability. However, these devices have complex architectures that are laborious to create and are prone to failure due to defects or dust. Generating libraries containing thousands of different droplets without sacrificing cost, quality, or scalability remained an open problem in the field.
Stage of Research
The inventors have created a new approach that allows for the creation of emulsified droplet libraries with high combinatorial diversity. First, each well is loaded with a unique reagent, templating particles, and surfactant-laden oil. This method uses particle-templated emulsification (PTE) to create droplets by breaking a continuous aqueous phase into consecutively smaller droplets by common methods of agitation used in labs (vortexing, pipetting, etc.). Hydrogel beads can be used as the template particles, which ensures that the agitation step will not cause the droplets to emulsify in sizes significantly smaller than that of the hydrogel beads. Additionally, because each sample is emulsified in its own tube, the production of emulsions can be scaled up by PTE via parallelization in plates. The emulsions in each separate well in the plates are then combined to create the droplet library. These libraries can then be sorted using standard microfluidic devices.
Applications
- Scalable creation of droplet libraries with high combinatorial diversity without sacrificing droplet emulsification quality.
- Digital droplet PCR can be performed after sorting to identify nucleic acid barcodes
- Method can be used for combinatorial drug screening and synthesis
Advantages
- PTE creates libraries with 30 seconds of plate vortexing that are compatible with microfluidics devices used for sorting.
- Libraries are monodisperse, making them compatible with microfluidics devices used for sorting
- Approach requires minimal equipment and expertise and is independent of the number and geometry of wells
Stage of Development
Research- in vivo
Publications
PCT/US2023/010423
Keywords
scRNA-seq, emulsification, high throughput screening, digital droplet PCR
Technology Reference
CZ Biohub ref. no. CZB-223F; UCSF ref. no. SF2022-018
