Poster Y9 - Microfluidic devices for exposing cell spheroids to chemical gradients

Introduction: Biomolecules, such as nutrients, signaling molecules, and drugs transported in the bloodstream permeate vessel walls and create concentration gradients in tissues through advection and diffusion. Various cell-derived cytokines also form local gradients and contribute to biological functions. For example, a gradient of vascular endothelial growth factor (VEGF), a cytokine derived from vascular endothelial cells, is involved in neovascularization in and around tumors, resulting in tumor growth and invasion. It is difficult to artificially control chemical gradients in vivo and mimic these gradients in vitro using conventional culture equipment and tools. This study developed a microfluidic device that generates chemical gradients in the cell culture layer and applied it to expose tumor cell spheroids under multiple chemical gradients.

Materials and

Methods: The microfluidic device consists of a spheroid layer and a 12 × 20 mm fluid layer (Figs. 1a and b). The spheroid layer has multiple o-ring-shaped wells (diameter: 1.2 mm) along the chemical gradient to prevent the downstream migration of spheroids by the fluid (Fig. 1c). Both layers were fabricated with polydimethylsiloxane (PDMS), and the layers and glass substrate were bonded through plasma treatment. The perfusion of a medium containing different concentrations of molecules from the two inlets formed chemical gradients in the spheroid layer. The chemical gradients were simulated in COMSOL, and the actual chemical gradients were quantified from the intensity of the rhodamine 6G. Tumor spheroids were prepared by culturing 2 × 10^4 HeLa cells with gelatin hydrogel-fragmented fibers (Genocel, NIKKE) for 4 days in 96-well round-bottom plates with an ultralow attachment surface. Hoechst 33342 and calcein-AM were used for nuclear and cytoplasmic staining, respectively, and were injected from two inlets at 30 μL/min for 2 h, and three spheroids were exposed under dye gradients.

Results, Conclusions, and Discussions: The chemical gradients quantified from the fluorescence intensities were in close agreement with the simulated values. As shown in Fig. 2, HeLa cell spheroids fluoresced according to the calcein-AM and Hoechst gradients, and the spheroid in the center of the fluid layer was counterstained. Chemical gradients can be easily controlled by arranging the molecular concentrations, culture layer structure, and flow rates, allowing for the formation of chemical gradients that mimic organ- and cell-specific microenvironments. A microfluidic cell culture device that generates an oxygen gradient was previously developed. Internal and surrounding tissues of tumors are partially, heterogeneously, and severely hypoxic, and exposing spheroids to a combination of oxygen and chemical gradients artificially mimics and controls the tumor microenvironment in vitro.
In this study, a microfluidic device that generates chemical gradients and exposes tumor spheroids to fluorescent dye gradients was developed. The proposed microfluidic device is expected to be applied not only to reproduce the tumor microenvironment but also to understand the molecular dose dependency in organoids that mimic various organs.

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