Robust Dielectrophoretic Cell Aggregation in Biocompatible Hydrogels

The ability to build a three-dimensional (3D) cellular construct is a field of increasing interest. Current cell-based in vitro studies are typically two-dimensional (2D) monolayer models that have been shown to be unreliable and non-predictive in a clinical setting  as they do not represent human tissue models [1-2].  In vitro 3D model development has been brought into focus in order to develop a system that can resemble the natural living tissue, allowing cells to assume their natural shape and interaction without the need for animal models. In comparison to 2D models, 3D cell culture allows cells to assume their natural shape, and allows cell-cell interactions which can affect disease progression and drug responses in cells [2].  Disease progression and drug efficacy have been shown to vary between 2D versus 3D constructs [3].  There is also debate whether the mechanism underpinning this increase in drug resistance is due to changes in the behaviour of cells due to cell-to-cell contact, or simply that the inner cells in a 3D construct are shielded from the drug due to the outer cells preventing diffusion of the drug across the whole cell mass.

Proposed systems for 3D spheroids (most flexible and well characterized in vitro model) formation such as spinners, shakers, rotaries, as well as hanging drop method are simple to use and designed for mass fabrication. These however, have difficulties maintaining a uniform size and geometry in micro-scale spheroids which are crucial when investigating avascular effects in cell constructs [4-6].  The use of dielectrophoresis (DEP) to construct hemispherical cell clusters in polymer hydrogels have shown promise [6-8] however clinical application of this technology, such as drug efficacy has yet to be shown.  A common method of representing this drug efficacy is the half maximal inhibitory concentration (IC₅₀) which measures the amount or dose of drug that is able to inhibit a specific biological process (such as cell division leading to cell death).  MTT is the current method of obtaining the IC₅₀ of a drug on a cell line grown in traditional monolayer culture. Since this method relies on cell suspensions, other methods must be used on 3D aggregates.

In this work we present an on-chip DEP device as a robust, high through-put, reproducible technique of cell aggregation and assessment of drug effectiveness. Using “dot” electrodes, we successfully aggregated and maintained 3D culture for several cell lines including yeast, k562 human leukaemia cells, cardiomyoctyes, and HeLa's. Further, the effect of Amphotericin B on patterned yeast cells and doxorubicin on the survival and proliferation of patterned and encapsulated cancer cell aggregates were observed. Comparisons of the 2D versus 3D IC₅₀ of these agents were investigated. In demonstrating DEP as a robust technique for cell aggregation, alternative hydrogels such as collagen and PuraMatrix™ were also investigated to provide a more biocompatible method of cell aggregation and to allow further study of aggregates through dissociation. The DEP dot electrodes have demonstrated the potential for rapid bench top cell aggregate formation and have shown promise in clinical application

.

References:

1.   Elliot.N.T and Yuan.F, A Review of Three-Dimensional In Vitro Tissue Models for Drug Discovery and Transport Studies. Journal of Pharmaceutical Sciences, 2010. 100(1): p. 59-74.

2.   Khetan.S and Burdick.J.A, Patterning hydrogels in three dimensions towards controlling cellular interactions. Soft Matter, 2011. 7: p. 830-838.

3.   Petersen.O.W, et al., Interaction with basement membrane serves to rapidly distinguish growth and differentiation pattern of normal and malignant human breast epithelial cells. Proceedings of the National Academy of Sciences of the United States of America, 1992. 89(19): p. 9064-9068.

4.   Hye-Jin Jin,   Young-Ho Cho,   Jin-Mo Gu,   Jhingook Kim and   Yong-Soo Oh,  A multicellular spheroid formation and extraction chip using removable cell trapping barriers. Lab chip, 2010, 11,115-119

5.   Heike Hardelauf,   Jean-Philippe Frimat,   Joanna D. Stewart,   Wiebke Schormann,   Ya-Yu Chiang,   Peter Lampen,   Joachim Franzke, Jan G. Hengstler   Cristina Cadenas,   Leoni A. Kunz-Schughart and   Jonathan West, Microarrays for the scalable production of metabolically relevant tumour spheroids: a tool for modulating chemosensitivity traits,  Lab Chip, 2011, 11, 419–428

6.   Rula G. Abdallat, Aziela S. Ahmad Tajuddin, David H. Gould, Michael P. Hughes, Henry O. Fatoyinbo, Fatima H. Labeed, 2013. Process development for cell aggregate arrays encapsulated in a synthetic hydrogel using negative dielectrophoresis. Electrophoresis volume 34 pp 1059-1067.

7.   Albrecht, D. R., Underhill, G. H., Mendelson, A. & Bhatia, S. N., 2007. Multiphase electropatterning of cells and biomaterials. The Royal Society of Chemistry, Volume 7, pp. 702-709.

8.   Agarwal S, Sebastian A, Forrester LM, Markx G. Formation of embryoid bodies using dielectrophoresis. Biomicrofluidics. 2012  Vol. 6, No. 2, 024101, 03.04.2012.

Figure  SEQ Figure \* ARABIC 1: HeLa cells aggregated on dot electrodes at time 0, and viability tested 48 hr post DEP exposure