Rapid Generation of Multiplexed Cell Cocultures Using Acoustic Droplet Ejection Followed by Aqueous Two-Phase Exclusion Patterning

Abstract

The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells.

Cell Co-culture Patterning Using Aqueous Two-phase Systems

“…liquid handling robots and acoustic droplet ejection), as presented in our previous studies 14,15,20 . It is also possible to generate DEX droplets that are much smaller in volume by pneumatically ejecting DEX through capillary orifices or by actuating an orifice in a microchannel to produce flowing DEX droplets sheathed by PEG 19 .…”

“…ATPSs formed by PEG and DEX generally display interfacial tensions that are much lower than other liquid-liquid two-phase systems such as oil and water; however, the interfacial tension forces still exert effects on small particles such as viruses, cells and protein aggregates 2,[11][12][13] . Finally, since higher molecular weight PEG and DEX separate at low concentrations (less than 5% wt/wt for high molecular weight polymers varieties) in the presence of physiological concentrations of salts, there are few if any deleterious effects on mammalian cells incorporated within these systems [14][15][16] . Recently, the interfacial properties and partitioning effects of ATPSs have been applied by our lab for cell patterning 14,[16][17][18][19][20] .…”

“…Finally, since higher molecular weight PEG and DEX separate at low concentrations (less than 5% wt/wt for high molecular weight polymers varieties) in the presence of physiological concentrations of salts, there are few if any deleterious effects on mammalian cells incorporated within these systems [14][15][16] . Recently, the interfacial properties and partitioning effects of ATPSs have been applied by our lab for cell patterning 14,[16][17][18][19][20] . This was accomplished by micropatterning a denser DEX solution on cell culture substrates in the presence of PEG.…”

A Perspective on 3D Bioprinting Technology: Present and Future

“…An inkjet-based printer is a non-contact printing technique that places extremely small droplets of ink on the substrate (Xu et al, 2013;Abeyewickreme et al, 2009;Fang et al, 2012). Inkjet printing could be further divided in to continuous or drop-on-demand methodology to create cellular constructs in a desired manner.…”

“…There is no droplet generation; instead a continuous stream of bio-inks is dispensed on the substrate. The printer setup normally consists of an orifice made of syringes, a motor that drives the syringes in 3-axis motion and a stage on which the object is constructed (Fang et al, 2012). For extrusion printing of cells, the material usually consists of a highly viscous cell-laden hydrogel that can flow from the nozzle without the need for high temperatures (Fedorovich et al, 2007).…”