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 ...

The University of Akron

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.

A robust polynomial fitting approach for contact angle measurements.

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.

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

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.

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

Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.

Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture.

This paper presents a new 3D culture microtechnology for high throughput production of tumor spheroids and validates its utility for screening anti-cancer drugs. We use two immiscible polymeric aqueous solutions and microprint a submicroliter drop of the "patterning" phase containing cells into a bath of the "immersion" phase. Selecting proper formulations of biphasic systems using a panel of biocompatible polymers results in the formation of a round drop that confines cells to facilitate spontaneous formation of a spheroid without any external stimuli. Adapting this approach to robotic tools enables straightforward generation and maintenance of spheroids of well-defined size in standard microwell plates and biochemical analysis of spheroids in situ, which is not possible with existing techniques for spheroid culture. To enable high throughput screening, we establish a phase diagram to identify minimum cell densities within specific volumes of the patterning drop to result in a single spheroid. Spheroids show normal growth over long-term incubation and dose-dependent decrease in cellular viability when treated with drug compounds, but present significant resistance compared to monolayer cultures. The unprecedented ease of implementing this microtechnology and its robust performance will benefit high throughput studies of drug screening against cancer cells with physiologically-relevant 3D tumor models.

Ehsan Atefi

Department of Biomedical Engineering

The University of Akron

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

Ehsan Atefi

.css-o250i3{font-size:18px;font-family:Open Sans,sans-serif;line-height:21.6px;}Department of Biomedical Engineering

The University of Akron

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

Department of Biomedical Engineering