Fabrication of Designable and Suspended Microfibers via Low-Voltage 3D Micropatterning

Building two-dimensional (2D) and three-dimensional (3D) fibrous structures in the micro- and nanoscale will offer exciting prospects for numerous applications spanning from sensors to energy storage and tissue engineering scaffolds. Electrospinning is a well-suited technique for drawing micro- to nanoscale fibers, but current methods of building electrospun fibers in 3D are restrictive in terms of printed height, design of macroscopic fiber networks, and choice of polymer. Here, we combine low-voltage electrospinning and additive manufacturing as a method to pattern layers of suspended mesofibers. Layers of fibers are suspended between 3D-printed supports in situ in multiple fiber layers and designable orientations. We examine the key working parameters to attain a threshold for fiber suspension, use those behavioral observations to establish a “fiber suspension indicator”, and demonstrate its utility through design of intricate suspended fiber architectures. Individual fibers produced by this method approach the micrometer/submicrometer scale, while the overall suspended 3D fiber architecture can span over a centimeter in height. We demonstrate an application of suspended fiber architectures in 3D cell culture, utilizing patterned fiber topography to guide the assembly of suspended high-cellular-density structures. The solution-based fiber suspension patterning process we report offers a unique competence in patterning soft polymers, including extracellular matrix-like materials, in a high resolution and aspect ratio. The platform could thus offer new design and manufacturing capabilities of devices and functional products by incorporating functional fibrous elements.


Section I -Tool Head Design
A controllable distance between the electrospinning syringe needle and support structure surface is crucial to facilitating the LEP process. The ability to calibrate the distance between the tip of any given nozzle, in this case the electrospinning syringe, and the build platform was an instrumental part of the printer's firmware customization. This allows flexibility in the length syringe tip used so printing can take place upon the grounded build plate, a glass slide or other elevated surfaces. Regular disassembly and reassembly of the electrospinning toolhead is also necessary to install fresh solution with the correct working parameters and to avoid nozzle clogging. This design requirement, in addition to the necessity in this configuration to switch tool-heads for alternating between different printing processes introduces a lot of scope for tool-head offsets in between mounting the different tools. Such offsets may prevent prints from having the appropriate precision and repeatability to enable this process. To address this, robust syringe loading and tool-head fastening on the toolmount is essential. Figure S1(a) used three ball-in-vee groove features, along with a supplementary cone feature and magnets to provide a fastening to the tool-mount. This provides a kinematic coupling that is statically determinate, constraining all 6 degrees of freedom Figure S1(b).

Our design
The picking up mechanism is magnetic and the drop-off mechanism utilises the shearing force against fitted slots in the docking stations. This design is shown to be able to accommodate a large degree of misalignment, allowing the tool-head change to function even when the tool-head has moved around in the tool dock. Inside the electrospinning tool-head (see S1(c)), a vee-groove was also used to provide two of the point contact forces, while a third force was provided by a grub screw. This creates a determinate arrangement in two dimensions (i.e. it constrains the 3 degrees of freedom in 2D), and the use of a second grub S-3 screw constrains the other degrees of freedom to maintain the distance between the syringe tip and printing substrate.
The robustness of the tool-head design was evaluated with time-lapse imaging and image analysis as illustrated in S2(a). Displacement of the electrospinning syringe needle from a reference object was measured in X, Y and Z to evaluate the design performance, as shown in S2(b) and summarized in Table S1. The largest deviation from the mean position was in the Y axis. Even when the syringe was reassembled, the mean deviation of the syringe tip in Z was 7.5 μm which is acceptable precision to ensure reliable LEP processing. Overall, the design enables a tolerance of up to 9 mm, should the tool heads be misaligned in the tool dock. This tolerance provides 3D LEP with adequate robustness and precision without the need for further manipulation or interfering offset calibration.
To create multiple layers of fibers, it is important to ensure that subsequent PLA deposition do not damage the fibers already deposited. The default thermoplastic nozzle on an Ultimaker Where ω is the rate at which the oscillatory rheometry is conducted, or the rate at which the "ink" is being extruded through the nozzle.

S8 Biological Experiments-Live/Dead Imaging and Immunostaining
A z-stack was acquired for the LIVE/DEAD study consisting of 10 slices over a depth of 285 μm using a 10x objective.