Tunable Superhydrophobic Microwell Array Devices for High-Throughput Culture of 3D Cancer Models

3D cancer models offer higher physiological relevance than 2D cancer models as they are able to recapitulate tumor architecture with higher fidelity. Products to produce cancer spheroids, such as the Aggrewell800, are available commercially but rely on user coating with an anti-adherence solution, have difficulty forming tight spheroids in some cell lines, and provide only two microwell sizes. Cancer cells are known to cluster in circulation, conferring higher shear and treatment resistance, in addition to increased metastatic potential compared to single cells. Current methods in the literature to produce <i>in vitro</i> models of circulating tumor cell clusters or microemboli (CTM) are inconsistent and lack the ability to control cluster size. The goal of our study was to utilize microfabrication techniques to design a chip with tunable microwell array and a superhydrophobic surface at the bottom to achieve a device that can produce a range of 3D cancer models. Two different superhydrophobic array devices (SHArD) have been implemented to reliably culture a spheroid model (SHArD-S) and a CTM model (SHArD-C) <i>in vitro.</i><br/>Superhydrophobic surfaces were fabricated by spin coating a ZnO nanoparticle solution in MQ water onto a 3” Si wafer. Wafers were annealed and then baked at 90C for 4 hr while submerged in a zinc nitrate hexahydrate and HMTA bath to grow nanorods. ZnO nanorod thin film was characterized via SEM to be highly uniform and reproducible. The nanorods were randomly oriented on the wafer surface and had an average length of 500µm. Width ranged from 40nm to 100nm, corresponding to the size of the initial nanoparticle powder. Ultrathick SU8 lithography was performed for the SHArD-S to obtain a microwell wall height of about 400μm with an aspect ratio of 3. Lines were spaced 800μm apart resulting in square sections of 650x650μm². For the SHArD-C, the adhesion promoter OmniCoat was spin coated onto the wafer surface. SU8 lithography was done to create a 75µm thick grid, resulting in 100 x 100µm² microwells. The final step for both devices was to deposit a layer of non-adherent polymer via C₄F₈ plasma polymerization in an Oxford PlasmaPro100. Coating was confirmed to be homogenous and maintain surface nanoroughness via SEM and water contact angle (WCA) measurements. The static WCA of the nanorods coated with the non-adherent polymer was 167°. The dynamic WCA were 165.2° and 168.6° for the advancing WCA and receding WCA, respectively. Microfabrication processes and characterization were carried out at the VINSE cleanroom and the CNMS cleanroom at Oak Ridge National Laboratories.<br/>For spheroid formation, the colorectal cancer cell line HCT116 was plated for 4 days with in the microwells at 2.5k, 5k, and 7.5k cells per microwell in the SHArD-S and the Aggrewell800 for comparison. Spheroids were observed via confocal microscopy by staining with DAPI to quantify compactness. Spheroids cultured in the SHArD-S were significantly less round and more compact than those cultured in the Aggrewell800, more closely resembling tumors in the body. Additionally, spheroids were exposed to physiologically relevant fluid shear stress (FSS) of 188 s^-1 using a cone-and-plate viscometer to assess cell:cell adhesion stability. Spheroids grown in the Aggrewell 800 plates displayed a large decrease in cross-sectional area after FSS, while those grown in the SHArD-S showed no significant change in cross-sectional area. For CTM formation, HCT116 cells were plated for 48hrs in the SHArD-C at 3, 5, and 7 cells per microwell. Brightfield microscopy showed that CTM size was easily controlled by varying plating density of the cells within each microwell. CTMs grown in the SHArD-C did not disaggregate when exposed to FSS in a cone-and-plate viscometer. We thus provide a novel method to reliably produce highly tunable 3D cancer models from cancer CTMs to cancer spheroids, allowing one to simulate cell:cell interactions throughout the metastatic cascade.