Scalable Fabrication and Use of 3D Structured Microparticles Spatially Functionalized with Biomolecules

Abstract

Microparticles with defined shapes and spatial chemical modification can enable new opportunities to interface with cells and tissues at the cellular scale. However, conventional methods to fabricate shaped microparticles have trade-offs between the throughput of manufacture and precision of particle shape and chemical functionalization. Here, we achieved scalable production of hydrogel microparticles at rates of greater than 40 million/hour with localized surface chemistry using a parallelized step emulsification device and temperature-induced phase-

Sorting single T cells based on secreted cytokines and surface markers using hydrogel nanovials

“…Our nanovial technology can be easily scaled up to process millions of cells per day. Here, we fabricated nanovials using a flow-focusing device at rate of 5.6 million/hour, and higher production rates can be achieved using parallelized microfluidic device 17 . For each secretion assay, we loaded 0.23 million cells into 187,000 nanovials in a 24-well plate yielding ~28,000 cells loaded in nanovials.…”

“…with in vitro or animal models) or transcriptomic information (either pooled or using single-cell sequencing technologies). In our previous studies, we have shown that the morphology of the nanovial cavity protects cells during damaging sorting steps, leading to improved viability, cell expansion and proliferation post-sort 16,17 . The ability to functionally sort viable intact cells will allow improved understanding of unique polyfunctional phenotypes.…”

“…Oil partitioned the aqueous phases into monodisperse water-in-oil droplets and PEG-gelatin polymers phase separated after approximately 5 seconds, followed by cross-linking with focused UV light through a DAPI filter set and microscope objective (Nikon, Eclipse Ti-S) near the outlet region of the microfluidic device. Polymerized nanovials were collected in a conical tube and any unreacted phases including oil were removed through a series of washing steps as previously described 16,17 . Biotinylation of the gelatin-layer formed in the nanovial cavity was conducted by incubating nanovials with 10 mM Sulfo-NHS-Biotin (APExBIO) overnight at 4°C.…”

“…Using an aqueous two-phase system combined with droplet microfluidics and hydrogel chemistry, we fabricated cavity-containing hydrogel microparticles. With some modifications on our lab's previously reported study 16,17 , we used a flow-focusing device to generate millions of monodisperse polyethylene glycol (PEG)-based nanovials with the inner cavity selectively coated with biotinylated gelatin (Figure S1.A). To accommodate human T cells with diameters of ~10 μm we fabricated uniform nanovials with an average diameter of 35 μm (CV = 5.1%) and average cavity of 21.2 μm (CV = 7.2%).…”

“…We analyzed secretion signal by gating on the green fluorescence signal on nanovials and found less than 1% of gated nanovials without cells also appeared in our positive secretion gate during the 3 hour activation period (Figure 4C), suggesting minimal cross-talk. Previously, we have seen limited crosstalk in gelatin-functionalized nanovials where the cavity is selectively conjugated with capture antibodies 16,17 . Presumably, secreted cytokines accumulate at higher concentrations locally and are captured but when cytokines diffuse or are advected to neighboring cavities the concentration is diluted substantially leading to a reduced signal.…”