Dynamic 3D Printing With In Situ Depolarization: A New Biomanufacturing Paradigm for Guided Cell-Cell Communication

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In an electrohydrodynamics (EHD)-based additive manufacturing (AM) process, an applied voltage pulls a cylindrical jet of polymer material from a needle to a collector plate. The cylindrical jet forms a deposited fiber that acquires a characteristic charge. Conventional EHD processes enable direct printing of materials at small scales, but are hampered by limited accuracy and pattern control of the deposited charged fibers. To address this limitation, this research focuses on the fundamental understanding of fiber charge effects observed in EHD processes. It is hypothesized that restoring a neutral fiber charge will enable fibers to be precisely deposited. In the absence of a net charge, the fibers can be aligned and layered to produce a 3D biological substrate for cells to attach and function. Specifically, this fundamental knowledge will yield a biological substrate seeded with rat brain neural cells and endothelial cells. In this two-cell culture platform, the morphology of the neural cells are directed by the deposited fiber architecture to form physical contacts with the endothelial cells. The results of this work can have a significant impact on the life sciences by furnishing robust 3D cell culture platforms for drug screening and fundamental cell studies. Moreover, the AM methodology can be adapted for precision pattern control of polymer fiber structures across a wide range of industries, including electronics and medicine. The research outcomes will be integrated into the undergraduate curriculum as well as introduce high school students to advanced manufacturing, and broaden participation of underrepresented groups in research.The overall goal of this research is EHD-based additive manufacturing of biological substrates composed of discharged fibers. Such fibers can be reliably oriented in prescribed directions and positions to spatially guide cell morphology. Currently, the residual charge entrapped within successively deposited EHD fibers yields electrostatic forces between fibers that constrain the printed pattern resolution. The first research objective is to understand the effects of print surface temperature on the residual fiber charge. A PID-controlled thermoelectric printbed will be constructed to allow fundamental investigations regarding thermal depolarization of the printed fibers to a neutral charge state. A picoammeter will be integrated to enable in-process fiber charge measurements. Temperature and time-resolved charge measurements will be correlated to quantify the charge decay phenomena. Fiber placement accuracy will be measured as a ratio of inter-fiber distance to fiber diameter using scanning electron microscopy. The second research objective is to test the effect of dynamic fiber placement accuracy on prescribed cell-cell contacts in a two-cell culture model of rat brain neural cells and endothelial cells. To accomplish this objective, the discharged fiber surface will be patterned with growth factors along the layering direction. These growth factors will stimulate the neural cells to project extensions that form contacts with endothelial cells on the topmost layer. Immunochemistry of cell surface markers and barrier permeability measurements will be conducted to confirm cell-cell communication between the rat brain neural and endothelial cells.