Research Thrust 3:

Single Cell Array Assembly Using Liquid Microdroplets 


In single-cell analysis, cellular activities are assayed on an individual, rather than population-average basis. This provides a complete profile of the cellular status of the cell population to reflect the heterogeneity within the population. Thus, trapping and patterning a large number of cells are crucial for bio-printing, cell-cell interaction studies, and drug development with single cell analysis. For instance, constraining the location of cells according to defined positions allows for the prolonged visualization of individual cellular development, potentially in the presence of modifying reagents. This provides an opportunity to observe and analyze phenomena that are normally averaged out in bulk cell culture. Despite these advantages, patterning a large number of cells with single cell resolution is still challenging. Most techniques to date rely on physical forces to relocate single cells to desired positions or mechanical structures to constrain single cells in these positions. These methods pose significant damage to the cells’ overall health and significantly reduce their viability. In addition, the techniques cannot achieve the required throughput and the single cell resolution at the same time. 

To address this challenge, we developed a new technique, µACD, for the facile generation of large-area cell arrays. This procedure relied on the generation of cell-loaded aerosol microdroplets and the assembly of these cell-loaded microdroplets into well-defined, large-area arrays using the action of stretchable chemical patterns engineered to include appropriate extracellular matrices (ECM) surface functionality. The microdroplets were generated using a spray nozzle that enabled control of the volume/size of the droplets delivered to the surface (Figure 3a-b). Assembly of the cell-loaded microdroplets was conveniently achieved via mechanically-induced droplet coalescence on elastomeric substrates with engineered surface-wettability patterns and ECM (Figure 3c-e). Cell proliferation inside the patterned areas was observed after standard culture techniques for a 72-hour time period (Figure 3f-h). Staining and imaging can be carried out after a prolonged cell culture for various biological assays. We even showed the potentially of patterning and monitoring single cells over a 48-hour time period (Figure 3i). Collectively, these data show that we can use µACD to generate patterned cell colonies from assembled 3D microdroplets.

Thrust 3.tif


Single cell array patterning using microdroplets. An elastic substrate was pre-patterned with an array of hydrophilic circles, with diameters ranging from 80 µm to 300 µm. This substrate will be strained to a level of ε=1. A micro-nozzle delivers a mixture of cell solution to the strained elastic substrate (a). Injecting from the micro-nozzle, the mixture becomes microdroplets with diameters less than 50 µm (b). The droplets will coalesce to the hydrophilic regions after the substrate strain is released (c-e). Cells delivered alongside the droplets will be patterned to the hydrophilic region (f). They will start to proliferate by attaching to the matrix protein in these hydrophilic regions (g-h). Staining the nucleus with DAPI (blue) and actin filament (green) shows that cells established cell-substrate adhesion (g-h). The cell suspension can also be tuned to enable the patterning and monitoring of single cells (i). Scale bars: b, 100 µ; e, 100 µm; f, 100 µm; g, 200 µm; h, 50 µm; I, 50 µm.

Major Publications:

Perez‐Toralla, Karla, Angel Olivera‐Torres, Mark A. Rose, Amir Monemian Esfahani, Keerthana Reddy, Ruiguo Yang, and Stephen A. Morin. Facile Production of Large‐Area Cell Arrays Using Surface‐Assembled Microdroplets. Advanced Science 7, 15 (2020): 2000769:1-8.