The development of tools for patterning cocultures of cells is a fundamental interest among cell biologists and tissue engineers. Although a variety of systems exist for micropatterning cells, the methods used to generate cell micropatterns are often cumbersome and difficult to adapt for tissue engineering purposes. This study combines acoustic droplet ejection and aqueous two-phase system exclusion patterning to introduce a method for patterning cocultures of cells in multiplexed arrays. This new method uses focused acoustic radiation pressure to eject discrete droplets of uniform size from the surface of a dextran solution containing cells. The size of droplets is controlled by adjusting ultrasound parameters, such as pulse, duration, and amplitude. The ejected dextran droplets are captured on a cell culture substrate that is manipulated by a computer-controlled 3D positioning system according to predesigned patterns. Polyethylene glycol solution containing an additional cell type is then added to the culture dish to produce a two-phase system capable of depositing different types of cells around the initial pattern of cells. We demonstrate that our method can produce patterns of islands or lines with two or more cell types. Further, we demonstrate that patterns can be multiplexed for studies involving combinations of multiple cell types. This method offers a tool to transfer cell-containing samples in a contact-free, nozzle-less manner, avoiding sample cross-contamination. It can be used to pattern cell cocultures without complicated fabrication of culture substrates. These capabilities were used to examine the response of cancer cells to the presence of a ligand (CXCL12) secreted from surrounding cocultured cells.

Brain-on-chip (BOC) technology such as nanogrooves and microtunnel structures can advance in vitro neuronal models by providing a platform with better means to maintain, manipulate and analyze neuronal cell cultures. Specifically, nanogrooves have been shown to influence neuronal differentiation, notably the neurite length and neurite direction. Here, we have drawn new results from our experiments using both 2D and 3D neuronal cell culture implementing both flat and nanogrooved substrates. These are used to show a comparison between the number of cells and neurite length as a first indicator for valuable insights into baseline values and expectations that can be generated from these experiments toward design optimization and predictive value of the technology in our BOC toolbox. Also, as a new step toward neuronal cell models with multiple compartmentalized neuronal cell type regions, we fabricated microtunnel devices bonded to both flat and nanogrooved substrates to assess their compatibility with neuronal cell culture. Our results show that with the current experimental protocols using SH-SY5Y cells, we can expect 200 – 400 cells with a total neurite length of approximately 4,000–5,000 μm per 1 mm 2 within our BOC devices, with a lower total neurite length for 3D neuronal cell cultures on flat substrates only. There is a statistically significant difference in total neurite length between 2D cell culture on nanogrooved substrates versus 3D cell culture on flat substrates. As extension of our current BOC toolbox for which these indicative parameters would be used, the microtunnel devices show that culture of SH-SY5Y was feasible, though a limited number of neurites extended into microtunnels away from the cell bodies, regardless of using nanogrooved or flat substrates. This shows that the novel combination of microtunnel devices with nanogrooves can be implemented toward neuronal cell cultures, with future improvements to be performed to ensure neurites extend beyond the confines of the wells between the microtunnels. Overall, these results will aid toward creating more robust BOC platforms with improved predictive value. In turn, this can be used to better understand the brain and brain diseases.

We studied the micropattern fidelity of a Norland Optical Adhesive 81 (NOA81) microsieve made by soft-lithography and laser micromachining. Ablation opens replicated cavities, resulting in three-dimensional (3D) micropores. We previously demonstrated that microsieves can capture cells by passive pumping. Flow, capture yield, and cell survival depend on the control of the micropore geometry and must yield high reproducibility within the device and from device to device. We investigated the NOA81 film thickness, the laser pulse repetition rate, the number of pulses, and the beam focusing distance. For NOA81 films spin-coated between 600 and 1200 rpm, the pulse number controls the breaching of films to form the pore’s aperture and dominates the process. Pulse repetition rates between 50 and 200 Hz had no observable influence. We also explored laser focal plane to substrate distance to find the most effective ablation conditions. Scanning electron micrographs (SEM) of focused ion beam (FIB)-cut cross sections of the NOA81 micropores and inverted micropore copies in polydimethylsiloxane (PDMS) show a smooth surface topology with minimal debris. Our studies reveal that the combined process allows for a 3D micropore quality from device to device with a large enough process window for biological studies.

We demonstrated the microtransfer molding of Norland Optical Adhesive 81 (NOA81) thin films. NOA81 nanogrooves and flat thin films were transferred from a flexible polydimethylsiloxane (PDMS) working mold. In the case of nanogrooves, the mold's feature area of 15 × 15 mm2 contains a variety of pattern dimensions in a set of smaller nanogroove fields of a few mm2 each. We demonstrated that at least six microtransfers can be performed from the same PDMS working mold. Within the restriction of our atomic force microscopy measurement technique, nanogroove height varies with 82 ± 11 nm depending on the pattern dimensions of the measured fields. Respective micrographs of two of these fields, i.e., one field designated with narrower grooves (D1000L780, case 1) and the other designated with wider grooves (D1000L230, case 2) but with the same periodicity values, demonstrate faithful transfer of the patterns. The designated pattern dimensions refer to the periodicity (D) and the ridge width (L) in the original design process of the master mold (dimensional units are nm). In addition, neither NOA81 itself (flat films) nor NOA81 nanogroove thin films with a thickness of 1.6 μm deteriorate the imaging quality in optical cell microscopy.

We have investigated the laser micromachining of microsieves with 3D micropore geometries. We hypothesize that mechanical cues resulting from the positioning and machining of ablated holes inside a pyramidal microcavity can influence the direction of neuronal outgrowth and instruct stem cell-derived neural networks in their differentiation processes. We narrowed the number of variations in device fabrication by developing a numerical model to estimate the stress distribution in a cell interacting with the laser-tailored unique 3D geometry of a microsieve’s pore. Our model is composed of two components: a continuous component (consisting of the membrane, cytoplasm, and nucleus) and a tensegrity structural component (consisting of the cytoskeleton, nucleoskeleton, and intermediate filaments). The final values of the mechanical properties of the components are selected after evaluating the shape of the continuous cell model when a gravity load is applied and are compared to the shape of a cell on a glass substrate after 3 h. In addition, a physical criterion implying that the cell should not slip through a hole with a bottom aperture of 3.5  μm is also set as a constraint. Among all the possible one- or multi-hole configurations, six cases appeared promising in influencing the polarization process of the cell. These configurations were selected, fabricated, and characterized using scanning electron microscopy. Fabricated microsieves consist of a 20  μm thick Norland Optical Adhesive 81 (NOA81) foil with an array of inverted pyramidal microcavities, which are opened by means of KrF 248 nm laser ablation. By changing the position of the laser beam spot on the cavities (center, slope, or corner) as well as the direction of laser beam with respect to the NOA81 microcavity foil (top side or back side), different ablation configurations yielded a variety of geometries of the 3D micropores. In the one-hole configurations when the shot is from the top side, to make the desired diameter of 3.5  μm (or less) of an opening, 1500 laser pulses are sufficient for the center and slope openings. This requirement is around 2000 laser pulses when the aperture is positioned in the corner. In back side ablation processes, the required number of pulses for through-holes at the center, slope, and corner positions are 1200, 1800, and 1800 pulses, respectively. In conclusion, we developed a microsieve platform that allows us to tailor the 3D topography of individual micropores according to the selection of cases guided by our numerical stress distribution models.