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

[This corrects the article DOI: 10.3389/fchem.2019.00441.].

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

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 (NaHCO3, NaH2PO4, 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.

Precision cell delivery in biphasic polymer systems enhances growth of keratinocytes in culture and promotes their attachment on acellular dermal matrices.

Current approaches for precision deposition of cells are not optimized for moist environments or for substrates with complex surface topographic features, for example, the surface of dermal matrices and other biomaterials. To overcome these challenges, an approach is presented that utilizes cell confinement in phase-separating polymer solutions of polyethylene glycol and dextran to precisely deliver keratinocytes in well-defined colonies. Using this approach, keratinocyte colonies are produced with superior viability, proliferative capacity, and barrier formation compared with the same number of cells dispersedly seeded across substrate surfaces. It is further demonstrated that keratinocytes delivered in colonies to the surface of acellular dermal matrices form an intact epidermal basal layer more rapidly and more completely than cells delivered by conventional dispersed seeding. These findings demonstrate that delivery of keratinocytes in phase-separating polymer solutions holds potential for enhancing growth of keratinocytes in culture and production of functional skin equivalents.

Emerging Biotechnology Applications of Aqueous Two-Phase Systems.

Liquid-liquid phase separation between aqueous solutions containing two incompatible polymers, a polymer and a salt, or a polymer and a surfactant, has been exploited for a wide variety of biotechnology applications throughout the years. While many applications for aqueous two-phase systems fall within the realm of separation science, the ability to partition many different materials within these systems, coupled with recent advances in materials science and liquid handling, has allowed bioengineers to imagine new applications. This progress report provides an overview of the history and key properties of aqueous two-phase systems to lend context to how these materials have progressed to modern applications such as cellular micropatterning and bioprinting, high-throughput 3D tissue assembly, microscale biomolecular assay development, facilitation of cell separation and microcapsule production using microfluidic devices, and synthetic biology. Future directions and present limitations and design considerations of this adaptable and promising toolkit for biomolecule and cellular manipulation are further evaluated.

Microscopic evaluation of aqueous two-phase system emulsion characteristics enables rapid determination of critical polymer concentrations for solution micropatterning.

Aqueous two-phase systems have emerged as valuable tools for microscale analysis of cell growth and many other biotechnology applications. The most critical step in developing an aqueous two-phase system for a specific application is identifying the critical concentrations at which the polymer solutions phase-separate. Current techniques for determining these critical concentrations rely on laborious methods, highly specialized assays or computational methods that make this step difficult for non-specialists. To overcome these limitations, we present a simplified assay that uses only readily accessible laboratory instruments and consumables (e.g., multichannel micropipettes, 96-well plates and a simple compound microscope) to determine the critical concentrations of aqueous two-phase system-forming polymers. We demonstrate that formulations selected from phase diagrams that describe these critical concentrations can be applied for solution micropatterning of cells.