Ehsan Atefi

Ultralow interfacial tensions of aqueous two-phase systems measured using drop shape.

Aqueous solutions of different polymers can separate and form aqueous two-phase systems (ATPS). ATPS provide an aqueous, biocompatible, and mild environment for separation and fractionation of biomolecules. The interfacial tension between the two aqueous phases plays a major role in ATPS-mediated partition of biomolecules. Because of the structure of the two aqueous phases, the interfacial tensions between the phases can be 3-4 orders of magnitude smaller than conventional fluid-liquid systems: ∼1-100 μJ/m(2) for ATPS compared to ∼72 mJ/m(2) for the water-vapor interface. This poses a major challenge for the experimental measurements of reproducible interfacial tension data for these systems. We address the need for precise determination of ultralow interfacial tensions by systematically studying a series of polymeric ATPS comprising of polyethylene glycol (PEG) and dextran (DEX) as the phase-forming polymers. Sessile and pendant drops of the denser DEX phase are formed within the immersion PEG phase. An axisymmetric drop shape analysis (ADSA) is used to determine interfacial tensions of eight different ATPS. Specific criteria are used to reproducibly determine ultralow interfacial tensions of the ATPS from pendant and sessile drops. Importantly, for a given ATPS, pendant drop and sessile drop experiments return values within 0.001 mJ/m(2) indicating reliability of our measurements. Then, the pendant drop technique is used to measure interfacial tensions of all eight ATPS. Our measured values range from 0.012 ± 0.001 mJ/m(2) to 0.381 ± 0.006 mJ/m(2) and vary with the concentration of polymers in equilibrated phases of ATPS. Measurements of ultralow interfacial tensions with such reproducibility will broadly benefit studies involving partition of different biomolecules in ATPS and elucidate the critical effect of interfacial tension.

Interfacial Tension Effect on Cell Partition in Aqueous Two-Phase Systems.

Aqueous two-phase systems (ATPS) provide a mild environment for the partition and separation of cells. We report a combined experimental and theoretical study on the effect of interfacial tension of polymeric ATPS on the partitioning of cells between two phases and their interface. Two-phase systems are generated using polyethylene glycol and dextran of specific properties as phase-forming polymers and culture media as the solvent component. Ultralow interfacial tensions of the solutions are precisely measured using an axisymmetric drop shape analysis method. Partition experiments show that two-phase systems with an interfacial tension of 30 μJ/m(2) result in distribution of majority of cells to the bottom dextran phase. An increase in the interfacial tension results in a distribution of cells toward the interface. An independent cancer cell spheroid formation assay confirms these observations: a drop of the dextran phase containing cancer cells is dispensed into the immersion polyethylene glycol phase to form a cell-containing drop. Only at very small interfacial tensions do cells remain within the drop to aggregate into a spheroid. We perform a thermodynamic modeling of cell partition to determine variations of free energy associated with displacement of cells in ATPS with respect to the ultralow interfacial tensions. This modeling corroborates with the experimental results and demonstrates that at the smallest interfacial tension of 30 μJ/m(2), the free energy is a minimum with cells in the bottom phase. Increasing the interfacial tension shifts the minimum energy and partition of cells toward the interfacial region of the two aqueous phases. Examining differences in the partition behavior and minimum free energy modeling of A431.H9 cancer cells and mouse embryonic stem cells shows that the surface properties of cells further modulate partition in ATPS. This combined approach provides a fundamental understanding of interfacial tension role on cell partition in ATPS and a framework for future studies.

A Robust Polynomial Fitting Approach for Contact Angle Measurements

Polynomial fitting to drop profile offers an alternative to well-established drop shape techniques for contact angle measurements from sessile drops without a need for liquid physical properties. Here, we evaluate the accuracy of contact angles resulting from fitting polynomials of various orders to drop profiles in a Cartesian coordinate system, over a wide range of contact angles. We develop a differentiator mask to automatically find a range of required number of pixels from a drop profile over which a stable contact angle is obtained. The polynomial order that results in the longest stable regime and returns the lowest standard error and the highest correlation coefficient is selected to determine drop contact angles. We find that, unlike previous reports, a single polynomial order cannot be used to accurately estimate a wide range of contact angles and that a larger order polynomial is needed for drops with larger contact angles. Our method returns contact angles with an accuracy of <0.4° for solid-liquid systems with θ < ~60°. This compares well with the axisymmetric drop shape analysis-profile (ADSA-P) methodology results. Above about 60°, we observe significant deviations from ADSA-P results, most likely because a polynomial cannot trace the profile of drops with close-to-vertical and vertical segments. To overcome this limitation, we implement a new polynomial fitting scheme by transforming drop profiles into polar coordinate system. This eliminates the well-known problem with high curvature drops and enables estimating contact angles in a wide range with a fourth-order polynomial. We show that this approach returns dynamic contact angles with less than 0.7° error as compared to ADSA-P, for the solid-liquid systems tested. This new approach is a powerful alternative to drop shape techniques for estimating contact angles of drops regardless of drop symmetry and without a need for liquid properties.

Automated, spatio‐temporally controlled cell microprinting with polymeric aqueous biphasic system

Cell printing is a promising approach to create organized constructs for tissue engineering applications. We present an automated cell printing microtechnology based on the use of an aqueous two‐phase system (ATPS) interfaced with a three‐axis motorized system. Cells suspended in the denser aqueous dextran (DEX) phase are loaded into printing tips, which are placed onto the cartridge of the motorized system. Using a computer interface, tips are lowered in the vicinity of a biological surface maintained in the immersion, aqueous polyethylene glycol (PEG) phase to perform a horizontal motion, autonomously dispense their contents onto the surface, and retracted out of the PEG phase. The motorized ATPS technology allows precise spatial and temporal control of the printing process and supports printing fully viable cells. We conduct a systematic study and show that the resolution of ATPS‐mediated cellular patterns depends on several factors including the dimensions of the printing tips, lateral speed of tips during horizontal motion, and the loaded volume of the DEX phase in the tips. The finest resolution is ∼300 µm obtained with a tip diameter of 200 µm at a printing tip speed of 16.5 mm/s. Higher speeds result in unstable DEX patterns that break into drops due to capillary instability, and thus are avoided. We also test a number of printing substrates and find that in addition to a cell monolayer, decellularized matrices can serve as a substrate for cell printing with ATPS. Using the principles from the characterization studies, we create duplex prints of cells to demonstrate the potential of this approach for spatio‐temporally controlled cell placement. The ATPS printing microtechnology will be a step forward toward developing well‐organized, three‐dimensional tissue constructs. Biotechnol. Bioeng. 2014;111: 404–412.

Membrane Shape Instability Induced by Protein Crowding.

Peripheral proteins can bend membranes through several different mechanisms, including scaffolding, wedging, oligomerization, and crowding. The crowding effect in particular has received considerable attention recently, in part because it is a colligative mechanism-implying that it could, in principle, be explored by any peripheral protein. Here we sought to clarify to what extent this mechanism is exploited by endocytic accessory proteins. We quantitatively investigate membrane curvature generation by means of a GUV shape stability assay. We found that the amount of crowding required to induce membrane curvature is correlated with membrane tension. Importantly, we also revealed that at the same membrane tension, the crowding mechanism requires far higher protein coverage to induce curvature changes compared to those observed for the endophilin BAR domain, serving here as an example of an endocytic accessory protein. Our results are important for the design of membrane-targeted biosensors as well as the understanding of mechanisms of biological membrane shaping.

Robotic Production of Cancer Cell Spheroids with an Aqueous Two-phase System for Drug Testing

Cancer cell spheroids present a relevant in vitro model of avascular tumors for anti-cancer drug testing applications. A detailed protocol for producing both mono-culture and co-culture spheroids in a high throughput 96-well plate format is described in this work. This approach utilizes an aqueous two-phase system to confine cells into a drop of the denser aqueous phase immersed within the second aqueous phase. The drop rests on the well surface and keeps cells in close proximity to form a single spheroid. This technology has been adapted to a robotic liquid handler to produce size-controlled spheroids and expedite the process of spheroid production for compound screening applications. Spheroids treated with a clinically-used drug show reduced cell viability with increase in the drug dose. The use of a standard micro-well plate for spheroid generation makes it straightforward to analyze viability of cancer cells of drug-treated spheroids with a micro-plate reader. This technology is straightforward to implement both robotically and with other liquid handling tools such as manual pipettes.