Dielectrophoretic assembly of insulinoma cells and fluorescent nanosensors into three-dimensional pseudo-islet constructs

Abstract

Dielectrophoretic forces, generated by radio-frequency voltages applied to micromachined, transparent, indium tin oxide electrodes, have been used to condense suspensions of insulinoma cells (BETA-TC-6 and INS-1) into a 10x10 array of threedimensional cell constructs. Some of these constructs, measuring approximately 150 μm in diameter and 120 μm in height, and containing around 1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the dielectrophoretic force maintained, these engineered cell constructs were able to withstand mechanical shock and fluid flow forces. Reproducibility of the process required knowledge of cellular dielectric properties, in terms of membrane capacitance and membrane conductance, which were obtained by electrorotation measurements. The ability to incorporate fluorescent nanosensors, as probes of cellular oxygen and pH levels, into these 'pseudo-islets' was also demonstrated. The footprint of the 10x10 array of cell constructs was compatible with that of a 1536 microtitre plate, and thus amenable to optical interrogation using automated plate reading equipment.

Review Article—Dielectrophoresis: Status of the theory, technology, and applications

“…104 Electrode arrays have also been produced by the excimer laser ablation of indium tin oxide ͑ITO͒ electrodes. [105][106][107] Although the optical transparency of an ITO film is high, making this electrode material particularly suitable for observing DEP particle manipulation with transmission microscopy, UV radiation such as that used in an excimer laser beam is efficiently absorbed by the ITO for it to be micromachined.…”

“…Suspensions of insulinoma ͑beta͒ cells have been directed into 3D aggregates in an array of 10ϫ 10 field cages, using micromachined ITO electrodes and negative DEP. 107 Some of these beta-cell constructs, measuring approximately 150 m in diameter and 120 m in height and containing ϳ1000 cells, were of the same size and cell density as a typical islet of Langerhans. With the DEP force maintained, the cell constructs were able to withstand mechanical shock and fluid flow forces.…”

dc electrokinetic transport of cylindrical cells in straight microchannels

“…Experimental results and numerical predictions indicate that cylindrical particles are always aligned with their longest axis parallel to the electric field under high electric fields, which is to be used to align and assemble nanowires [44][45][46][47][48][49] and biological tissues. 63 In addition, a higher electric field leads to a faster particle alignment. Further numerical studies indicate that cylindrical particles can experience oscillatory motions under low electric fields.…”

Pseudoislets as primary islet replacements for research

“…13,14 These β-cell line pseudoislets show much improved insulin secretory characteristics compared to equivalent cells maintained as monolayers, demonstrating the importance of homotypic β-cell to β-cell interactions in the generation and maintenance of appropriate insulin secretory responses, and validating the pseudoislet model for in vitro studies of the regulation of insulin secretion. Since the first publication of the characteristics of MIN6 pseudoislets in 1999, 13 several other laboratories have adopted cell line-derived pseudoislet models for research, [15][16][17][18][19] but this model is still not widely used despite the advantages of widespread availability, simplicity and cost-effectiveness. The Diabetes Research Group at King's College London recently (April 2010) hosted a symposium with the aims of disseminating knowledge about the pseudoislet model and highlighting emerging research themes from current studies using pseudoislets.…”