[n]Cycloparaphenylenes as Compatible Fluorophores for Melt Electrowriting.

Fluorescent probes are an indispensable tool in the realm of bioimaging technologies, providing valuable insights into the assessment of biomaterial integrity and structural properties. However, incorporating fluorophores into scaffolds made from melt electrowriting (MEW) poses a challenge due to the sustained, elevated temperatures that this processing technique requires. In this context, [n]cycloparaphenylenes ([n]CPPs) serve as excellent fluorophores for MEW processing with the additional benefit of customizable emissions profiles with the same excitation wavelength. Three fluorescent blends are used with distinct [n]CPPs with emission wavelengths of either 466, 494, or 533 nm, identifying 0.01 wt% as the preferred concentration. It is discovered that [n]CPPs disperse well within poly(ε-caprolactone) (PCL) and maintain their fluorescence even after a week of continuous heating at 80 °C. The [n]CPP-PCL blends show no cytotoxicity and support counterstaining with commonly used DAPI (Ex/Em: 359 nm/457 nm), rhodamine- (Ex/Em: 542/565 nm), and fluorescein-tagged (Ex/Em: 490/515 nm) phalloidin stains. Using different color [n]CPP-PCL blends, different MEW fibers are sequentially deposited into a semi-woven scaffold and onto a solution electrospun membrane composed of [8]CPP-PCL as a contrasting substrate for the [10]CPP-PCL MEW fibers. In general, [n]CPPs are potent fluorophores for MEW, providing new imaging options for this technology.

Why bioprinting in regenerative medicine should adopt a rational technology readiness assessment.

Bioprinting is an annex of additive manufacturing, as defined by the American Society for Testing and Materials (ASTM) and International Organization for Standardization (ISO) standards, characterized by the automated deposition of living cells and biomaterials. The tissue engineering and regenerative medicine (TE&RM) community has eagerly adopted bioprinting, while review articles regularly herald its imminent translation to the clinic as functional tissues and organs. Here we argue that such proclamations are premature and counterproductive; they place emphasis on technological progress while typically ignoring the critical stage-gates that must be passed through to bring a technology to market. We suggest the technology readiness level (TRL) scale as a valuable metric for gauging the relative maturity of a bioprinting technology in relation to how it has passed a series of key milestones. We suggest guidelines for a bioprinting-oriented scale and use this to discuss the state-of-the-art of bioprinting in regenerative medicine (BRM) today. Finally, we make corresponding recommendations for improvements to BRM research that would support its progression to clinical translation.

Materials and Strategies to Enhance Melt Electrowriting Potential.

Melt electrowriting (MEW) is an emerging additive manufacturing (AM) technology that enables the precise deposition of continuous polymeric microfibers, allowing for the creation of high-resolution constructs. In recent years, MEW has undergone a revolution, with the introduction of active properties or additional functionalities through novel polymer processing strategies, the incorporation of functional fillers, postprocessing, or the combination with other techniques. While extensively explored in biomedical applications, MEW's potential in other fields remains untapped. Thus, this review explores MEW's characteristics from a materials science perspective, emphasizing the diverse range of materials and composites processed by this technique and their current and potential applications. Additionally, the prospects offered by postprinting processing techniques are explored, together with the synergy achieved by combining melt electrowriting with other manufacturing methods. By highlighting the untapped potentials of MEW, this review aims to inspire research groups across various fields to leverage this technology for innovative endeavors.

Bioassembly of hemoglobin-loaded photopolymerizable spheroids alleviates hypoxia-induced cell death.

The delivery of oxygen within tissue engineered constructs is essential for cell survivability; however, achieving this within larger biofabricated constructs poses a significant challenge. Efforts to overcome this limitation often involve the delivery of synthetic oxygen generating compounds. The application of some of these compounds is problematic for the biofabrication of living tissues due to inherent issues such as cytotoxicity, hyperoxia and limited structural stability due to oxygen inhibition of radical-based crosslinking processes. This study aims to develop an oxygen delivering system relying on natural-derived components which are cytocompatible, allow for photopolymerization and advanced biofabrication processes, and improve cell survivability under hypoxia (1% O2). We explore the binding of human hemoglobin (Hb) as a natural oxygen deposit within photopolymerizable allylated gelatin (GelAGE) hydrogels through the spontaneous complex formation of Hb with negatively charged biomolecules (heparin, hyaluronic acid, and bovine serum albumin). We systematically study the effect of biomolecule inclusion on cytotoxicity, hydrogel network properties, Hb incorporation efficiency, oxygen carrying capacity, cell viability, and compatibility with 3D-bioassembly processes within melt electrowritten (MEW) scaffolds. All biomolecules were successfully incorporated within GelAGE hydrogels, displaying controllable mechanical properties and cytocompatibility. Results demonstrated efficient and tailorable Hb incorporation within GelAGE-Heparin hydrogels. The developed system was compatible with microfluidics and photopolymerization processes, allowing for the production of GelAGE-Heparin-Hb spheres. Hb-loaded spheres were assembled into MEW polycaprolactone scaffolds, significantly increasing the local oxygen levels. Ultimately, cells within Hb-loaded constructs demonstrated good cell survivability under hypoxia. Taken together, we successfully developed a hydrogel system that retains Hb as a natural oxygen deposit post-photopolymerization, protecting Hb from free-radical oxidation while remaining compatible with biofabrication of large constructs. The developed GelAGE-Heparin-Hb system allows for physoxic oxygen delivery and thus possesses a vast potential for use across broad tissue engineering and biofabrication strategies to help eliminate cell death due to hypoxia.

Cryo-Electrohydrodynamic Jetting of Aqueous Silk Fibroin Solutions.

The incorporation of 3D-printing principles with electrohydrodynamic (EHD) jetting provides a harmonious balance between resolution and processing speed, allowing for the creation of high-resolution centimeter-scale constructs. Typically, EHD jetting of polymer melts offers the advantage of rapid solidification, while processing polymer solutions requires solvent evaporation to transition into solid fibers, creating challenges for reliable printing. This study navigates a hybrid approach aimed at minimizing printing instabilities by combining viscous solutions and achieving rapid solidification through freezing. Our method introduces and fully describes a modified open-source 3D printer equipped with a frozen collector that operates at -35 °C. As a proof of concept, highly concentrated silk fibroin aqueous solutions are processed into stable micrometer scale jets, which rapidly solidify upon contact with the frozen collector. This results in the formation of uniform microfibers characterized by an average diameter of 27 ± 5 µm, a textured surface, and porous internal channels. The absence of instabilities and the notably fast direct writing speed of 42 mm·s-1 enable precise, fast, and reliable deposition of these fibers into porous constructs spanning several centimeters. The effectiveness of this approach is demonstrated by the consistent production of biologically relevant scaffolds that can be customized with varying pore sizes and shapes. The achieved degree of control over micrometric jet solidification and deposition dynamics represents a significant advancement in EHD jetting, particularly within the domain of aqueous polymer solutions, offering new opportunities for the development of intricate and functional biological structures.

Melt Electrowriting of Nylon-12 Microfibers with an Open-Source 3D Printer.

This study demonstrates how either a heated flat or cylindrical collector enables defect-free melt electrowriting (MEW) of complex geometries from high melting temperature polymers. The open-source "MEWron" printer uses nylon-12 filament and combined with a heated flat or cylindrical collector, produces well-defined fibers with diameters ranging from 33 ± 4 to 95 ± 3 µm. Processing parameters for stable jet formation and minimal defects based on COMSOL thermal modeling for hardware design are optimized. The balance of processing temperature and collector temperature is achieved to achieve auxetic patterns, while showing that annealing nylon-12 tubes significantly alters their mechanical properties. The samples exhibit varied pore sizes and wall thicknesses influenced by jet dynamics and fiber bridging. Tensile testing shows nylon-12 tubes are notably stronger than poly(ε-caprolactone) ones and while annealing has limited impact on tensile strength, yield, and elastic modulus, it dramatically reduces elongation. The equipment described and material used broadens MEW applications for high melting point polymers and highlights the importance of cooling dynamics for reproducible samples.

Technical improvements in preparing 3D printed anatomical models for comminuted fracture preoperative planning.

Preoperative planning of comminuted fracture repair using 3D printed anatomical models is enabling surgeons to visualize and simulate the fracture reduction processes before surgery. However, the preparation of such models can be challenging due to the complexity of certain fractures, particularly in preserving fine detail in bone fragments, maintaining the positioning of displaced fragments, and accurate positioning of multiple bones. This study described several key technical considerations for preparing 3D printed anatomical models for comminuted fracture preoperative planning. An optimized segmentation protocol was developed that preserves fine detail in bone fragments, resulting in a more accurate representation of the fracture. Additionally, struts were manually added to the digital model to maintain the positioning of displaced fragments after fabrication, reducing the likelihood of errors during printing or misrepresentation of fragment positioning. Magnets were also used to enable separation and visualization of accurate positioning of multiple bones, making it easier to visualize fracture components otherwise obscured by the anatomy. Finally, the infill for non-target structures was adjusted to minimize print time and material wastage. These technical optimizations improved the accuracy and efficiency of preparing 3D printed anatomical models for comminuted fracture preoperative planning, improving opportunities for surgeons to better plan surgical treatment in advance, reducing the likelihood of errors, with the goal of improving surgical outcomes.

Function Follows Form: Oriented Substrate Nanotopography Overrides Neurite-Repulsive Schwann Cell-Astrocyte Barrier Formation in an In Vitro Model of Glial Scarring.

Schwann cell (SC) transplantation represents a promising therapeutic approach for traumatic spinal cord injury but is frustrated by barrier formation, preventing cell migration, and axonal regeneration at the interface between grafted SCs and reactive resident astrocytes (ACs). Although regenerating axons successfully extend into SC grafts, only a few cross the SC-AC interface to re-enter lesioned neuropil. To date, research has focused on identifying and modifying the molecular mechanisms underlying such scarring cell-cell interactions, while the influence of substrate topography remains largely unexplored. Using a recently modified cell confrontation assay to model SC-AC barrier formation in vitro, highly oriented poly(ε-caprolactone) nanofibers were observed to reduce AC reactivity, induce extensive oriented intermingling between SCs and ACs, and ultimately enable substantial neurite outgrowth from the SC compartment into the AC territory. It is anticipated that these findings will have important implications for the future design of biomaterial-based scaffolds for nervous tissue repair.

A Decade of Melt Electrowriting.

Over the past decade, melt electrowriting (MEW) has established the fundamental understanding of processing (and printer) requirements. Iterative work on parametric development and dissemination of this recent additive manufacturing technology has been performed across many systems and polymers (mainly poly-(ε-caprolactone)), showing similarities and trends. However, the software and hardware ecosystems of MEW are not mature. Further, due to its multi-parametric nature, MEW can be challenging for laboratories to master. This review intends to provide a unique perspective on the dynamic relationship between MEW processing parameters. Such parameters can be divided into 1) those that affect the polymer flow rate to or 2) from the nozzle, and 3) environmental conditions. The most influential parameters for high-quality printing are applied voltage, applied pressure, collector speed, polymer temperature, nozzle diameter, and the conditions that lead to charge buildup (e.g., relative humidity). Other factors such as ambient temperature, nozzle size, and protrusion, collector temperature and conductivity, and collector distance can all affect the process. Success for MEW printing means fibers fall onto the collector according to their pre-programmed path with predicted fiber diameter. Here, the authors elucidate how the dynamic relationship between these parameters can converge into ideal printing conditions to produce scaffolds.

Primary Glial Cell and Glioblastoma Morphology in Cocultures Depends on Scaffold Design and Hydrogel Composition.

3D cell cultures better replicate the in vivo environment compared to 2D models. Glioblastoma multiforme, a malignant brain tumor, highly profits from its cellular environment. Here, the U87 glioblastoma cell line in the presence/absence of primary astrocytes is studied. Thiolated hyaluronic acid (HA-SH) hydrogel reinforced with microfiber scaffolds is compared to Matrigel. Hyaluronic acid is a major extracellular matrix (ECM) component in the brain. Poly(ɛ-caprolactone) (PCL) scaffolds are written by meltelectrowriting in a box and triangular shaped design with pore sizes of 200 µm. Scaffolds are composed of 10-layers of PCL microfibers. It is found that scaffold design has an impact on cellular morphology in the absence of hydrogel. Moreover, the used hydrogels have profound influences on cellular morphology resulting in spheroid formation in HA-SH for both the tumor-derived cell line and astrocytes, while cell viability is high. Although cocultures of U87 and astrocytes exhibit cell-cell interactions, polynucleated spheroid formation is still present for U87 cells in HA-SH. Locally restricted ECM production or inability to secrete ECM proteins may underlie the observed cell morphologies. Thus, the 3D reinforced PCL-HA-SH composite with glioma-like cells and astrocytes constitutes a reproducible system to further investigate the impact of hydrogel modifications on cellular behavior and development.

Electrohydrodynamic 3D Printing of Aqueous Solutions.

Among the various electrohydrodynamic (EHD) processing techniques, electrowriting (EW) produces the most complex 3D structures. Aqueous solution EW similarly retains the potential for additive manufacturing well-resolved 3D structures, while providing new opportunities for processing biologically derived polymers and eschewing organic solvents. However, research on aqueous-based EHD processing is still limited. To summarize the field and advocate for increased use of aqueous bio-based materials, this review summarizes the most significant contributions of aqueous solution processing. Special emphasis has been placed on understanding the effects of different printing parameters, the prospects for 3D processing new materials, and future challenges.

Reinforced Hyaluronic Acid-Based Matrices Promote 3D Neuronal Network Formation.

3D neuronal cultures attempt to better replicate the in vivo environment to study neurological/neurodegenerative diseases compared to 2D models. A challenge to establish 3D neuron culture models is the low elastic modulus (30-500 Pa) of the native brain. Here, an ultra-soft matrix based on thiolated hyaluronic acid (HA-SH) reinforced with a microfiber frame is formulated and used. Hyaluronic acid represents an essential component of the brain extracellular matrix (ECM). Box-shaped frames with a microfiber spacing of 200 µm composed of 10-layers of poly(ɛ-caprolactone) (PCL) microfibers (9.7 ± 0.2 µm) made via melt electrowriting (MEW) are used to reinforce the HA-SH matrix which has an elastic modulus of 95 Pa. The neuronal viability is low in pure HA-SH matrix, however, when astrocytes are pre-seeded below this reinforced construct, they significantly support neuronal survival, network formation quantified by neurite length, and neuronal firing shown by Ca2+ imaging. The astrocyte-seeded HA-SH matrix is able to match the neuronal viability to the level of Matrigel, a gold standard matrix for neuronal culture for over two decades. Thus, this 3D MEW frame reinforced HA-SH composite with neurons and astrocytes constitutes a reliable and reproducible system to further study brain diseases.

Prevascularized Retrievable Hybrid Implant to Enhance Function of Subcutaneous Encapsulated Islets.

Replacement of pancreatic ß-cells is one of the most promising treatment options for treatment of type 1 diabetes (T1D), even though, toxic immunosuppressive drugs are required. In this study, we aim to deliver allogeneic ß-cell therapies without antirejection drugs using a bioengineered hybrid device that contains microencapsulated ß-cells inside 3D polycaprolactone (PCL) scaffolds printed using melt electrospin writing (MEW). Mouse ß-cell (MIN6) pseudoislets and QS mouse islets are encapsulated in alginate microcapsules, without affecting viability and insulin secretion. Microencapsulated MIN6 cells are then seeded within 3D MEW scaffolds, and these hybrid devices implanted subcutaneously in streptozotocin-treated diabetic NOD/SCID and BALB/c mice. Similar to NOD/SCID mice, blood glucose levels (BGL) are lowered from 30.1 to 4.8 mM in 25-41 days in BALB/c. In contrast, microencapsulated islets placed in prevascularized MEW scaffold 3 weeks after implantation in BALB/c mice normalize BGL (<12 mM) more rapidly, lasting for 60-105 days. The lowering of glucose levels is confirmed by an intraperitoneal glucose tolerance test. Vascularity within the implanted grafts is demonstrated and quantified by 3D-doppler ultrasound, with a linear increase over 4 weeks (r = 0.65). Examination of the device at 5 weeks shows inflammatory infiltrates of neutrophils, macrophages, and B-lymphocytes on the MEW scaffolds, but not on microcapsules, which have infrequent profibrotic walling. In conclusion, we demonstrate the fabrication of an implantable and retrievable hybrid device for vascularization and enhancing the survival of encapsulated islets implanted subcutaneously in an allotransplantation setting without immunosuppression. This study provides proof-of-concept for the application of such devices for human use, but, will require modifications to allow translation to people with T1D. Impact statement The retrievable 3D printed PCL scaffold we have produced promotes vascularization when implanted subcutaneously and allows seeded microencapsulated insulin-producing cells to normalize blood glucose of diabetic mice for at least 2 months, without the need for antirejection drugs to be administered. The scaffold is scalable for possible human use, but will require modification to ensure that normalization of blood glucose levels can be maintained long term.

The Multiweek Thermal Stability of Medical-Grade Poly(ε-caprolactone) During Melt Electrowriting.

Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that places unique constraints on the processing of thermally degradable polymers. With a single nozzle, MEW operates at low throughput and in this study, medical-grade poly(ε-caprolactone) (PCL) is heated for 25 d at three different temperatures (75, 85, and 95 °C), collecting daily samples. There is an initial increase in the fiber diameter and decrease in the jet speed over the first 5 d, then the MEW process remains stable for the 75 and 85 °C groups. When the collector speed is fixed to a value at least 10% above the jet speed, the diameter remains constant for 25 d at 75 °C and only increases with time for 85 and 95 °C. Fiber fusion at increased layer height is observed for 85 and 95 °C, while the surface morphology of single fibers remain similar for all temperatures. The properties of the prints are assessed with no observable changes in the degree of crystallinity or the Young's modulus, while the yield strength decreases in later phases only for 95 °C. After the initial 5-d period, the MEW processing of PCL at 75 °C is extraordinarily stable with overall fiber diameters averaging 13.5 ± 1.0 µm over the entire 25-d period.

Brain and Breast Cancer Cells with PTEN Loss of Function Reveal Enhanced Durotaxis and RHOB Dependent Amoeboid Migration Utilizing 3D Scaffolds and Aligned Microfiber Tracts.

BACKGROUND: Glioblastoma multiforme (GBM) and metastatic triple-negative breast cancer (TNBC) with PTEN mutations often lead to brain dissemination with poor patient outcome, thus new therapeutic targets are needed. To understand signaling, controlling the dynamics and mechanics of brain tumor cell migration, we implemented GBM and TNBC cell lines and designed 3D aligned microfibers and scaffolds mimicking brain structures. METHODS: 3D microfibers and scaffolds were printed using melt electrowriting. GBM and TNBC cell lines with opposing PTEN genotypes were analyzed with RHO-ROCK-PTEN inhibitors and PTEN rescue using live-cell imaging. RNA-sequencing and qPCR of tumor cells in 3D with microfibers were performed, while scanning electron microscopy and confocal microscopy addressed cell morphology. RESULTS: In contrast to the PTEN wildtype, GBM and TNBC cells with PTEN loss of function yielded enhanced durotaxis, topotaxis, adhesion, amoeboid migration on 3D microfibers and significant high RHOB expression. Functional studies concerning RHOB-ROCK-PTEN signaling confirmed the essential role for the above cellular processes. CONCLUSIONS: This study demonstrates a significant role of the PTEN genotype and RHOB expression for durotaxis, adhesion and migration dependent on 3D. GBM and TNBC cells with PTEN loss of function have an affinity for stiff brain structures promoting metastasis. 3D microfibers represent an important tool to model brain metastasizing tumor cells, where RHO-inhibitors could play an essential role for improved therapy.

An initiator- and catalyst-free hydrogel coating process for 3D printed medical-grade poly(ε-caprolactone).

Additive manufacturing or 3D printing as an umbrella term for various materials processing methods has distinct advantages over many other processing methods, including the ability to generate highly complex shapes and designs. However, the performance of any produced part not only depends on the material used and its shape, but is also critically dependent on its surface properties. Important features, such as wetting or fouling, critically depend mainly on the immediate surface energy. To gain control over the surface chemistry post-processing modifications are generally necessary, since it's not a feature of additive manufacturing. Here, we report on the use of initiator and catalyst-free photografting and photopolymerization for the hydrophilic modification of microfiber scaffolds obtained from hydrophobic medical-grade poly(ε-caprolactone) via melt-electrowriting. Contact angle measurements and Raman spectroscopy confirms the formation of a more hydrophilic coating of poly(2-hydroxyethyl methacrylate). Apart from surface modification, we also observe bulk polymerization, which is expected for this method, and currently limits the controllability of this procedure.

Spinal Cord Neuronal Network Formation in a 3D Printed Reinforced Matrix-A Model System to Study Disease Mechanisms.

3D cell cultures allow a better mimicry of the biological and mechanical environment of cells in vivo compared to 2D cultures. However, 3D cell cultures have been challenging for ultrasoft tissues such as the brain. The present study uses a microfiber reinforcement approach combining mouse primary spinal cord neurons in Matrigel with melt electrowritten (MEW) frames. Within these 3D constructs, neuronal network development is followed for 21 days in vitro. To evaluate neuronal development in 3D constructs, the maturation of inhibitory glycinergic synapses is analyzed using protein expression, the complex mechanical properties by assessing nonlinearity, conditioning, and stress relaxation, and calcium imaging as readouts. Following adaptation to the 3D matrix-frame, mature inhibitory synapse formation is faster than in 2D demonstrated by a steep increase in glycine receptor expression between days 3 and 10. The 3D expression pattern of marker proteins at the inhibitory synapse and the mechanical properties resemble the situation in native spinal cord tissue. Moreover, 3D spinal cord neuronal networks exhibit intensive neuronal activity after 14 days in culture. The spinal cord cell culture model using ultrasoft matrix reinforced by MEW fibers provides a promising tool to study and understand biomechanical mechanisms in health and disease.

A novel in vitro assay for peripheral nerve-related cell migration that preserves both extracellular matrix-derived molecular cues and nanofiber-derived topography.

BACKGROUND: Molecular composition and topography of the extracellular matrix (ECM) influence regenerative cell migration following peripheral nerve injury (PNI). Advanced tissue engineering strategies for the repair of neurotmesis-type PNI include the development of nanofiber-containing implantable scaffolds that mimic features of the ECM to orchestrate regenerative growth. Reliable and quantifiable in vitro assays are required to assess the ability of such substrates to influence migration of the cell types of interest. However, most popular migration assays monitor cell migration into a cell exclusion zone (CEZ) but have dubious abilities to preserve the molecular and topographical cues of the substrate. NEW METHOD: Elastic band spacers (EBS), a simple, economical and standardized technique for the generation of well-defined CEZ based on the use of commercially available elastic bands, are introduced. RESULTS: EBS could sufficiently preserve ECM-derived molecular and poly(ε-caprolactone) (PCL) nanofiber-derived topographical cues. The application of EBS in the absence and presence of nanofiber-derived topographical cues was validated using perineurial cells and Schwann cells, both known to play key roles in peripheral nerve regeneration. COMPARISON WITH EXISTING METHODS: In contrast to EBS, commercial silicone inserts and the popular scratch assay caused substantial ECM substrate disruption, thereby preventing these techniques from being included in further investigations employing deposition of PCL nanofibers and cell migration analysis. CONCLUSIONS: EBS represent a useful addition to the existing repertoire of migration assays offering significant benefits in terms of substrate preservation. The simplicity and economy of the approach make it immediately accessible to research groups at minimal extra expense.

Convergence of Machine Vision and Melt Electrowriting.

Melt electrowriting (MEW) is a high-resolution additive manufacturing technology that balances multiple parametric variables to arrive at a stable fabrication process. The better understanding of this balance is underscored here using high-resolution camera vision of jet stability profiles in different electrical fields. Complementing this visual information are fiber-diameter measurements obtained at precise points, allowing the correlation to electrified jet properties. Two process signatures-the jet angle and for the first time, the Taylor cone area-are monitored and analyzed with a machine vision system, while SEM imaging for diameter measurement correlates real-time information. This information, in turn, allows the detection and correction of fiber pulsing for accurate jet placement on the collector, and the in-process assessment of the fiber diameter. Improved process control is used to successfully fabricate collapsible MEW tubes; structures that require exceptional accuracy and printing stability. Using a precise winding angle of 60° and 300 layers, the resulting 12 mm-thick tubular structures have elastic snap-through instabilities associated with mechanical metamaterials. This study provides a detailed analysis of the fiber pulsing occurrence in MEW and highlights the importance of real-time monitoring of the Taylor cone volume to better understand, control, and predict printing instabilities.

Design of Suspended Melt Electrowritten Fiber Arrays for Schwann Cell Migration and Neurite Outgrowth.

In this study, well-defined, 3D arrays of air-suspended melt electrowritten fibers are made from medical grade poly(ɛ-caprolactone) (PCL). Low processing temperatures, lower voltages, lower ambient temperature, increased collector distance, and high collector speeds all aid to direct-write suspended fibers that can span gaps of several millimeters between support structures. Such processing parameters are quantitatively determined using a "wedge-design" melt electrowritten test frame to identify the conditions that increase the suspension probability of long-distance fibers. All the measured parameters impact the probability that a fiber is suspended over multimillimeter distances. The height of the suspended fibers can be controlled by a concurrently fabricated fiber wall and the 3D suspended PCL fiber arrays investigated with early post-natal mouse dorsal root ganglion explants. The resulting Schwann cell and neurite outgrowth extends substantial distances by 21 d, following the orientation of the suspended fibers and the supporting walls, often generating circular whorls of high density Schwann cells between the suspended fibers. This research provides a design perspective and the fundamental parametric basis for suspending individual melt electrowritten fibers into a form that facilitates cell culture.