Nanostructured Cell Culture Device for Modeling Circulating Tumor Cell Clusters

Introduction: Manipulation of the biological-material interface in mammalian cell culture may be used to control cellular adhesion to the substrate or to other cells. Surfaces designed to be hydrophobic easily adsorb extracellular matrix proteins and promote proliferation and cell adhesion to the substrate. Such surfaces have a water contact angle (WCA) over 90°. In comparison, hydrophilic (WCA < 90°) and superhydrophobic (WCA > 150°) surfaces are not adhesive to cells or bacteria. When in contact with cell culture media, most hydrophilic surfaces are not conducive to cell growth due to hydration; however, interaction with cells may still occur. Superhydrophobic surfaces, in contrast, create a self-cleaning surface which repels protein adsorption and blocks cell adhesion due to trapped air beneath the liquid phase, resulting in ultra-low contact areas. These devices, known as the SuperHydrophobic microwell Array Device – Clusters (SHArD-C), specifically present an opportunity for creation of high-throughput cultures of metastatic cancer aggregate models meant for behavioral, genetic, and drug response analysis.

Materials and

Methods: Silicon wafers were pre-cleaned in acetone, then plasma cleaned to promote surface interactions. A Zinc Oxide (ZnO) nanoparticle suspension was spin coated onto the wafer and then dried at room temperature (RT). Annealing was performed to improve the ZnO seed crystallinity followed by processing in an oven while submerged in a ZnO nanorod growth solution. Such growth enhanced surface roughness via extremely random growth patterns of rods away from the silicon surface. Photolithography patterns were used for the SHArD-C, complete with an array of 70 µm x 70 µm squares spaced 30 µm apart, comprising a 2x2 in pattern inside the 3 in silicon wafer. Wafers were diced to 11x11 mm squares and C4F8 plasma polymerized to create superhydrophobic devices capable of creating almost 12,000 cluster models per device. Scanning electron microscopy was used to characterize the resulting surfaces. Thin films were additionally characterized using x-ray diffraction before and after furnace annealing. Nanorod surface roughness before and after polymer coating was measured using atomic force microscopy, and SU-8 structure dimensions were measured using a profilometer. Finally, a goniometer was used to characterize the wettability of the device surface, with superhydrophobicity confirmed for up to six days while submerged in complete media.

Results, Conclusions, and Discussions: Fabrication of the SHArD-C is straightforward, easily scalable for larger quantities, requires only basic equipment found in most cleanrooms, and is designed to ensure highly reproducible results. First, ZnO thin films derived from solution were confirmed to be highly uniform, crystalline, and reproducible by SEM imaging (Fig. 1A) and x-ray diffraction (XRD) (Fig. 1B&C). After utilizing annealing as a structure/defect relaxation method, the loss of 68 two-theta peak – representing the (201) crystallographic plane – and increased average peak intensity was observed, indicating an increase in crystallinity within the material.

SEM imaging also revealed a uniformly sized, randomly oriented, rough nanorod layer on the wafer surface (Fig. 1D), all important design features for superhydrophobicity. Additionally, a goniometer was used to measure WCAs on anti-adherence coated polystyrene – a surface commonly used in 3D culturing devices relying on hydrophilicity, a polymerized-only silicon wafer, engineered SHArD-C before media submersion, and SHArD-C after a six-day submersion in fully supplemented cell culture media. WCA measurements were 6° for coated polystyrene, 115° for coated silicon wafer, 165.2° for pre-submersion SHArD-C, and 158° for post-submersion SHArD-C, respectively (Fig. 1E-G). These values demonstrate the superhydrophobicity achieved via nanoscale roughened surfaces with plasma polymerization and their quality maintenance over time periods beyond the required time for growing cell clusters.

The SHArD-C produces 12,100 model circulating tumor cell (CTC) clusters per single well of a 24-well plate. This far exceeds the number of 3D aggregates possible in a single well of AggreWell800, a commercially available approach, can produce (296 per well of a 24-well plate). Thus, the SHArD-C not only provides an optimized surface for 3D cell aggregation and growth, but also has a 41-fold increased capacity to produce cancer cell aggregates. Using an alternative method, such as a 96-well plate or hanging drop method, would further reduce the capacity for cluster production. For the first time, we have reported on a physiologically relevant CTC cluster model using superhydrophobic surfaces optimized for 3D cell culture. Such models may replace those which rely on hydrophilic surfaces known to yield looser, “flimsy” aggregates which yield low size uniformity between cell types.

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