Rehydration of Polymeric, Aqueous, Biphasic System Facilitates High Throughput Cell Exclusion Patterning for Cell Migration Studies

Abstract

This paper describes a cell-exclusion patterning method facilitated by a polymeric aqueous two-phase system. The immersion aqueous phase (polyethylene glycol) containing cells rehydrates a dried disk of the denser phase (dextran) on the substrate to form a dextran droplet. With the right properties of the phase-forming polymers, the rehydrating droplet remains immiscible with the immersion phase. Proper formulation of the two-phase system ensures that the interfacial tension between the rehydrating droplet and the surrounding aqueous phase prevents cells from crossing the interface so that cells only adhere to the regions of the substrate around the dextran phase droplet. Washing out the patterning two-phase reagents reveals a cell monolayer containing a well-defined circular gap that serves as the migration niche for cells of the monolayer. Migration of cells into the cell-excluded area is readily visualized and quantified over time. A 96-well plate format of this “gap healing” migration assay demonstrates the ability to detect inhibition of cell migration by known cytoskeleton targeting agents. This straightforward method, which only requires a conventional liquid handler and readily prepared polymer solutions, opens new opportunities for high throughput cell migration assays.

Cell Co-culture Patterning Using Aqueous Two-phase Systems

“…This method allows any researcher with access to a typical cell culture lab (access to a hood, CO 2 incubator, and micropipettes) and the aforementioned polymers to reproducibly pattern cells in monoculture and co-culture. Our lab has demonstrated this capability by printing arrays of cells to study cell migration in a wound healing assay and to examine the effects of juxtacrine and paracrine signaling in the differentiation of embryonic cells 16,17,20 . Other methods, including patterning of extracellular matrix 28 , inkjet printing 29 , and patterning by laminar flow in microfluidic devices 25,26 have also been used to localize cells.…”

“…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 .…”

“…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.…”

“…This was accomplished by micropatterning a denser DEX solution on cell culture substrates in the presence of PEG. When cells are incorporated into the PEG phase, they are excluded from entering the DEX droplets due to PEG/DEX interfacial tension 20 . When cells are patterned in the DEX phase, they are retained at the surface of the cell culture substrate by interfacial tension and partitioning 16,17,19 .…”

Quantitative analysis of bacterial preference for cancer secreting proteins

“…Cell migration plays essential roles in angiogenesis [l, 2], embryonic development [3], wound healing [4][5][6], and cancer metastasis [7] and it is often meditated by chemical gradient. [8] Until so far, many types of devices including microfluidic platform have been developed for cell migration study based on chemical gradients.…”