High-Throughput 3D Neural Cell Culture Analysis Facilitated by Aqueous Two-Phase Systems
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High-Throughput 3D Neural Cell Culture Analysis Facilitated by Aqueous Two-Phase Systems Kristin Robin Ko1, Rishima Agarwal1 and John Frampton1 1 School of Biomedical Engineering, 5981 University Ave, Halifax, NS B3H 4R2, Canada ABSTRACT The three-dimensional (3D) culture of neural cells in extracellular matrix (ECM) gels holds promise for modeling neurodegenerative diseases and pre-clinical evaluation of novel therapeutics. However, most current strategies for fabricating 3D neural cell cultures are not well suited to automated production and analysis. Here, we present a facile, replicable, 3D cell culture system that is compatible with standard laboratory equipment and high-throughput workflows. This system uses aqueous two-phase systems (ATPSs) to confine small volumes (5 and 10 µl) of a commonly used ECM hydrogel (Matrigel) into thin, discrete layers, enabling highly-uniform production of 3D neural cell cultures in a 96-well plate format. These 3D neural cell cultures can be readily analyzed by epifluorescence microscopy and microplate reader. Our preliminary results show that many common polymers used in ATPSs interfere with Matrigel gelation and instead form fibrous precipitates. However, 0.5% hydroxypropyl methylcellulose (HPMC) and 2.5% dextran 10 kDa (D10) were observed to retain Matrigel integrity and had minimal impact on cell viability. This novel system offers a promising yet accessible platform for highthroughput fabrication of 3D neural tissues using readily available and cost-effective materials. INTRODUCTION Understanding the complex cell-cell and cell-matrix interactions underlying brain physiology is key to developing improved treatments for neurodegenerative diseases. Animal models and two-dimensional (2D) cell cultures have been used for decades to study these interactions, but lab animals are not always reliable models for human disease and most 2D culture systems provide unnatural growth environments that can induce inappropriate cell morphology, differentiation and physiological function [1-2]. High drug development attrition rates in the field of neurology suggest that alternative methods will be necessary to obtain results that are more translatable to humans [3]. Three-dimensional (3D) cell culture has potential to improve pre-clinical evaluation of novel therapeutics by recapitulating properties of the brain microenvironment, e.g., mass transport characteristics, cell-cell interactions, and environmental cues. Matrigel, which is an extracellular matrix-based (ECM-based) hydrogel that self-assembles into an optically-clear scaffold at temperatures above 10 °C, offers a potential 3D platform that replicates some of these important properties. However, the neuroscience community and the pharmaceutical industry have been slow to adopt hydrogel systems such as Matrigel due to their relative complexity and incompatibility with standard high-throughput equipment, assays, and imaging platforms [4]. For example, although Matrigel can efficiently generate 3D cell-laden gels, air-liquid interfacial
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