Assay Development and Screening
Microparticles with defined 3D shapes and spatially-tailored chemical functionality enable new opportunities to automate biotechnology at the scale of cells and tissues. The current approach to manufacture these shaped particles, including bowl-shaped hydrogel particles that act as “nanovials” for single-cell assays, requires precise injection of multiple polymer precursors into flow focusing microfluidic geometries, limiting scalability. Further, these previous approaches lacked the ability to spatially pattern different chemical groups, limiting the flexibility of the platform.
We demonstrate a new induced-phase separation concept to overcome tradeoffs between particle complexity and fabrication throughput for the manufacture of microparticles with tunable localized surface chemistry and shape. We fabricate monodisperse 3D-axisymmetric particles with a chemically-functionalized cavity (bowl-shaped nanovials) at rates of greater than 40 million/hour using a parallelized step emulsification device and temperature-induced phase separation. The resulting particles are spatially patterned with gelatin, which enables selective single-cell attachment in the cavity of particles and local capture of secreted molecules.
The localized gelatin on the cavity promoted deterministic attachment of cells and could be spatially functionalized with affinity reagents, enabling single-cell secretion assays for production of antibody therapeutics without emulsification steps. We found that for the same cell seeding concentrations, nanovials with localized gelatin showed significantly lower multiplet fraction (~ 1% of cell-containing nanovials) as compared to uniformly distributed binding moieties ( > 80 % of cell-containing nanovials). Cells bound to localized gelatin-nanovials showed high viability ( >80 % over 5 days of culture). Also, the functionalized cavities facilitate adherent cell growth, acting as a carrier for cells to prevent cell death during standard assays that can induce high fluid dynamic shear stresses such as fluorescence activated cell sorting (FACS). When FACS-sorting adherent CHO cells bound to gelatin nanovials (~270 events/second), we found significantly higher viability than for freely-suspended cells (80.0% vs. 54.9%, p < 0.0001).
We further exploit this localization effect to selectively functionalize biotin and capture antibodies in the cavities of the particles enabling single-cell secretion assays with reduced crosstalk. To characterize the effect of localized capture antibodies on cross-talk, we measured the secretion of a human immunoglobin G (IgG) against interleukin 8 (IL-8) produced by CHO cells loaded on nanovials. Compared to nanovial functionalized uniformly with capture antibodies, our engineered nanovials with localized capture antibodies possessed a higher secretion signal and lower background intensity (p < 0.1), indicating that the localized capture antibody in the cavity of nanovials enriched the secretion signals and reduced secretion spread from cells to neighboring empty nanovials. Millions of cavity-functionalized nanovials can be used as tiny test tubes for automating and scaling single-cell functional assays using flow cytometry-based analysis and sorting.