Poster Y18 - On the Design and Fabrication of Nanoliter Volume Hanging Drop Networks for Spheroid Culture

Introduction: Hanging drop cultures provide a favorable environment for gentle, gel-free formation of highly uniform three-dimensional cell cultures. Generated tissues are often used in drug screening applications. Initial cell numbers can be limited, as with primary cells provided by minimally invasive biopsies. Therefore, it can be beneficial to divide cells into miniaturized arrays of hanging drops to provide a larger number of samples. To replenish cell culture media and execute assays in these arrays, it is necessary to introduce microfluidic channels.
Previous demonstrations of connected hanging drop arrays have been at the millimeter scale, and few have included continuous perfusion. To design truly microscale networks, the physical operation governed by the Laplace pressure of individual drops must be well understood (Fig. 1a). System stability, particularly under pressure perturbations, must be considered. We have developed a broadly applicable analytical model that predicts the behavior of microscale hanging drops based on drop geometric parameters, array size, and flow conditions. Based on the results of this model, we have designed and fabricated a connected hanging drop array with a characteristic size of 300 µm that includes drop pinning features and addressable groups of wells. The microfabrication process in silicon and glass yields transparent devices with micron-scale features. We produced a fluidic system and experimental apparatus suitable for on-chip cell culture with minimal environmental perturbations. In this report, we demonstrate on-chip perfusion of these nanoliter volume hanging drop networks.

Materials and

Methods: An analytical model was developed based on treatment of the hanging drop as a nonlinear capacitor in an electronic-hydraulic analogy. A MATLAB solver was developed to determine well pressures in response to arbitrary inlet and outlet pressures.
A fabrication process was developed to produce silicon and glass devices suitable for wells throughout the micron size range (Fig 1b). Briefly, channels were etched to 10 µm depth in a silicon wafer. The wafer was dry oxidized and anodically bonded to a borofloat glass wafer to enclose the channels. Well and port features were patterned and etched entirely through the silicon wafer using deep reactive ion etching. Approximately 5-µm-deep trenches were etched to define the retaining features. After cleaning, a final 200-nm-thick oxide layer was deposited over the exposed silicon. The wafer was split into individual devices by snapping along etched borders. A custom 3D printed interface provided a connection to the device.
A pressure-based pump was used to provide distinct pressures in water reservoirs at the inlet and outlet. While maintaining the same pressure difference, the outlet pressure was varied, and the hanging drops were imaged using a bright-field microscope.

Results, Conclusions, and Discussions: A well size of 300 µm was chosen. To prevent the passage of cells, a channel height of 10 µm was chosen. From these geometric parameters and a target flow rate of approximately 1 µL/min, the developed analytical model was able to determine the maximum number of wells. Ultimately, a 12x16 array with six addressable groups was chosen to accommodate roughly 100,000 cells at 500 cells per drop. A fabrication protocol was developed and optimized (Fig. 1b). An optical image of the completed device is shown in Figure 1c, with the transparency of the device and layout of the channels visible. Scanning electron microscope images of the wells with connecting channels (Fig. 1d) and drop-pinning surface features (Fig. 1e) show key features.
To validate the performance of the device under flow, it was connected through the interface to the fluidic system controlled by pressure-based pumps at the inlet and outlet. By maintaining the same pressure difference across the device but varying the outlet pressure, the range of drop volumes was demonstrated (Fig. 1f). Corresponding modeled well volumes are shown in Figure 1g. The pressure drop due to the channel resistance is apparent from the changing drop shape. With decreasing outlet pressure, drops progress from nearly the maximum volume (first image) to a state where both convex and concave drops are present (third image) and finally to completely concave (fourth image) as predicted by our modeling.
We have designed and fabricated the first nanoliter volume hanging drop network for potential application in drug screening of tumor models. Future work will prioritize the demonstration of spheroid formation on-chip. Now that the design framework and fabrication protocol are in place, the chip design parameters can be further optimized for specific on-chip experiments. One major challenge is avoiding clogging during the bulk cell seeding procedure. In the event of a clog, the open system is susceptible to destabilization or evaporation of the cell culture medium. The network can be further optimized to mitigate clogging and provide improved resistance to perturbations.

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