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Aqueous two-phase systems (ATPSs) have numerous applications in separation science, and more recently, in bioassays enabled by the solution micropatterning of cells. The most frequently used ATPS in these applications is the polyethylene glycol (PEG)-dextran (Dex) system, as the polymers that form this ATPS have been extensively characterized in terms of their physicochemical properties. However, in addition to this well-known system, there exist many other ATPSs with properties that may be exploited to improve upon the PEG-dextran system for specific applications. One of these underexplored systems is the ATPS formed from PEG/polyethylene oxide (PEO) and albumin. In this article, we characterize the phase separation of PEG (35 kDa) and polyethylene oxide (PEO) (200, 900, and 4,000 kDa) with bovine serum albumin (BSA). We describe the microscopic emulsion behavior of these systems in the presence of NaCl and compounds (NaHCO 3 , NaH 2 PO 4 , and HEPES) commonly used in buffer solutions and cell culture media. We further demonstrate that PEG- and PEO-albumin systems can be used in place of the PEG-dextran system for confinement of suspension-cultured cells (Jurkat T cells and RPMI-8226 B cells). Cell viability and morphology are examined for various polymer formulations relative to the commonly used PEG 35 kDa-Dex 500 kDa system and polymer-free cell culture medium. In addition, we examine cell activation for various phase-separating medium components by measuring IL-2 and IL-6 secretion. We demonstrate that we can confine immune cells and cytokines in the PEG-BSA system, and that this system can be employed to screen immune responses by enzyme-linked immunospot (ELISpot) assay. This new system represents a promising ATPS formulation for applications where low levels of baseline cell activation are required, for instance, when culturing immune cells.

Confinement of Suspension-Cultured Cells in Polyethylene Glycol/Polyethylene Oxide-Albumin Aqueous Two-Phase Systems