(158h) High-Throughput Generation and Study of 3D Spheroids in a Thiol-Acrylate Hydrogel Scaffold Using a Droplet Microfluidic Trapping Array

Culturing cancer cells in a three-dimensional (3D) environment better recapitulates in vivo conditions including cell-to-cell interactions and mass transfer limitations of metabolites, oxygen, and drugs. Recent drug studies have suggested that a high rate of pre-clinical and clinical failures results from mass transfer limitations associated with drug entry into solid tumors that 2D model systems cannot predict. As such, there is a need to develop high-throughput platforms to generate and study 3D tumor spheroids for drug efficacy. Existing approaches for 3D cell culture include hanging droplet arrays and spinner flasks; however, these methods suffer from low throughput and significant heterogeneity in spheroid size and uniformity. Recently, microfluidic devices have become a popular alternative to rapidly generate 3D spheroids. A popular approach utilizes microwell arrays that have been modified to prevent cellular attachment and force cellular aggregation into 3D spheroids. These devices can rapidly generate a large number of spheroids; however, most microwell arrays cannot facilitate on-chip interrogation of 3D spheroids and suffer from disaggregation due to fluid shear stress. Similarly, the spheroids generated in the above methods suffer from significant heterogeneity since they are generated from 100-500 different cells. Droplet microfluidic devices offer a promising alternative to grow 3D spheroids from a small number (~1-10) of cancer cells to reduce spheroid heterogeneity while simultaneously allowing for on-chip interrogation and evaluation. The goal of this work was to incorporate a novel thiol-acrylate (TA) hydrogel scaffold into a microfluidic droplet trapping array to generate and study 3D spheroids. The TA hydrogel has been previously demonstrated to severe as an excellent scaffold for 3D cell culture that does not require external initiation factors such as UV light, temperature, or additional reagents. Three different overhead trapping arrays were fabricated with 70, 150, or 300 µm circular traps to study the effect of droplet size and cell seeding density on spheroid formation and growth in the TA hydrogel scaffold. The overhead trapping array consisted of 1400 traps and was capable of ~99% droplet trapping and ~50% cellular encapsulation. The TA hydrogel allowed for rapid (~30-40 min) polymerization of the scaffold followed by removal of the oil phase and replacement with complete media to initiate spheroid growth. The growth and viability of model breast cancer spheroids using MCF7 at 37°C was confirmed for up to four days under static conditions. However, after four days spheroid size stabilized suggesting the need for continuous infusion of fresh growth media. This study also identified that a minimum number of encapsulated cells (~4-6) is needed to generate a spheroid and that single encapsulated cells are less likely to growth into a full-blown spheroid. The findings from this study highlight an alternative approach into generating 3D spheroids incorporating an easy-to-use, inexpensive scaffold that can be used for high-throughput drug screening.