Streamlining the highly reproducible fabrication of fibrous biomedical specimens towards standardization and high throughput

Streamlining the highly reproducible fabrication of fibrous biomedical specimens towards standardization and high throughput | Research Square window.SnipcartSettings = { analytics: { enabled: false } }; (function() { var accessVector = localStorage.getItem('access_vector') || ''; window.dataLayer = window.dataLayer || []; if (accessVector) { window.dataLayer.push({ user: { profile: { profileInfo: { snid: accessVector } } } }); } })(); (function(w,d,s,l,i){w[l]=w[l]||[];w[l].push({'gtm.start':new Date().getTime(),event:'gtm.js'});var f=d.getElementsByTagName(s)[0],j=d.createElement(s),dl=l!='dataLayer'?'&l='+l:'';j.async=true;j.src='https://www.googletagmanager.com/gtm.js?id='+i+dl;f.parentNode.insertBefore(j,f);})(window,document,'script','dataLayer','GTM-K279D39R'); Browse Preprints In Review Journals COVID-19 Preprints AJE Video Bytes Research Tools Research Promotion AJE Professional Editing AJE Rubriq About Preprint Platform In Review Editorial Policies Our Team Advisory Board Help Center Sign In Submit a Preprint Cite Share Download PDF Article Streamlining the highly reproducible fabrication of fibrous biomedical specimens towards standardization and high throughput Gregor Lang, Zan Lamberger, Camilla Mussoni, Nicoletta Murenu, and 7 more This is a preprint; it has not been peer reviewed by a journal. https://doi.org/ 10.21203/rs.3.rs-4101827/v1 This work is licensed under a CC BY 4.0 License Status: Published Journal Publication published 14 Dec, 2024 Read the published version in Advanced Healthcare Materials → Version 1 posted You are reading this latest preprint version Abstract Nano- and micro-fiber-based scaffolds bear enormous potential for their use in cell culture and tissue engineering, since they mimic natural collagen structures and may thus serve as biomimetic adhesive substrates. They have, however, so far been restricted to small scale production in research labs with high batch-to-batch variation. They are commonly produced via electrospinning or melt electro-writing and their delicate nature poses obstacles in detachment, storage, and transportation. This study focuses on overcoming challenges in the high throughput production and practical handling, introducing new methods to reproducibly prepare such scaffolds suitable for quantitative cell culture applications. Attention is given to the seamless handling and transfer of samples without compromising structural integrity. Challenges in detaching fibers without damage as well as storage, and transport are addressed. Cell culture studies demonstrate the methodological advantages, emphasizing the potential for standardized testing and biological readouts of these fiber materials. The developed methods are applicable across various electrospinning and melt electro-writing approaches and can essentially contribute to their utilization in laboratory research and commercial applications. Health sciences/Medical research/Translational research Health sciences/Medical research/Preclinical research Biological sciences/Biological techniques/High-throughput screening Biological sciences/Biotechnology/Nanobiotechnology/Nanofabrication and nanopatterning Biological sciences/Biotechnology/Biomaterials Figures Figure 1 Figure 2 Figure 3 Figure 4 Figure 5 1. Introduction Electrostatic spinning methods, including electrospinning (ES) and melt electro-writing (MEW), have gained considerable attention in scientific research in recent decades. Their capability to create fiber diameters in the nano- and submicron ranges, along with the extensive diversity of synthetic and natural polymers available for use renders them particularly suitable for tissue engineering. 1 , 2 These methods facilitate the fabrication of biomimetic scaffolds resembling the fibrillar collagen structures present in natural tissues (50–500 nm). 3 Furthermore, ongoing research investigates their potential in other medical applications such as wound coverings, 4 drug release, 5 as well as in the development of lab- and organ-on-a-chip devices. 6 , 7 Additionally, electrostatic spinning methods prove highly suitable for diverse technical applications due to the high surface-to-volume ratio of thin fibers and their remarkable porosity. These applications span various fields, including filtration, 8 textiles, 9 and sensing. 10 , 11 Despite their substantial potential, notably in the pharmaceutical sector, the commercial implementation of these material systems remains limited. 12 , 13 However, it is anticipated that with advancing technological developments, their market for laboratory research and industrial production will witness substantial growth. 14 A recent review article by Ji et al. underscores the urgent need to devise innovative methods for scaling up nano-/micro-fiber materials while simultaneously preserving their quality and consistency. 15 Nadaf et al. also highlighted this crucial aspect, emphasizing that stability is the primary challenge when dealing with nanofibers. 16 Concurrently, there is a strong call to establish standards in processing and testing. The electrostatic spinning process itself is relatively simple, requiring only a high-voltage source, a needle pump, a collector, and a suitable polymer solution or melt. However, the processing of delicate fiber mats poses a significant challenge. Often, spinning occurs directly onto a grounded or oppositely charged collector plate, or for aligned fibers, onto a rotating cylinder. Subsequently, the fiber mats must be carefully peeled off for further preparation, a step that may lead to damage such as cracks, holes, or overstretching of the nonwoven membrane. In many applications, the fibers need to undergo chemical modifications or coatings for functionalization. 12 , 17 – 21 This necessitates the transfer of samples into various agents, introducing mechanical stress and potential damage. With the advancement of increasingly precise analytical methods, such as those for characterizing the surfaces of functionalized fibers, the demands for the quality of the required samples also increase. 22 Correlative methods, for example, require the examination of a single sample in various devices, where selected spots must be precisely locatable. Wang et al. identified sample preparation as the greatest challenge in this regard and concluded, that especially in the field of biomaterials, novel fixation procedures need to be developed to better preserve cell and material structures. 23 In particular, thin nanofiber mats tend to collapse and entangle in a humid state, resulting in deformation and damage. Often, samples must be cut to an appropriate size for further testing or application, a process that can also compromise sensitive structures. In the past, individual solutions have been applied to address these challenges, such as detaching the nonwovens submersed in ethanol/water mixtures, low adhesive substrates or utilizing coatings such as gelatine. 24 – 28 Additionally, the fabrication of thicker fiber mats has been employed to enhance handling. Inserts have been used to immobilize samples, although this can lead to damage during the clamping of the nonwovens and, like the other strategies is hardly scalable. In summary, it can be affirmed that, at present, there is no universally applicable procedure to ensure the quality and integrity of nanofiber mats for various processing and application possibilities while consistently and adequately preparing samples in sufficient quantities. Frequently, the decision must be made between expedited handling that may cause damage to the scaffolds and laborious, slow work aimed at preserving the structures. This choice has significant implications for the reproducibility of results. To address this issue, we developed a new workflow that is not only easily implementable, universally applicable, scalable, and cost-effective but also can be carried out with standard laboratory equipment or minimal investment costs. Applying a sacrificial film as a substrate material in combination with laser cutting and fused deposition modeling (FDM) resulted in high throughput production of electrospun and melt-electrowritten scaffolds, that are perfectly manageable for virtually any subsequent step, be it analytical or application-oriented. 2. Results 2.1. Basic workflow to harvest electrospun and electrowritten scaffolds To facilitate seamless removal of the electrospun or electrowritten scaffolds from their substrates after production, we used a water/ethanol soluble sacrificial cast PVA coating cast on steel plates or aluminium foil. The FDA-approved PVA is known to not adsorb to polymers that constitute the scaffolds, whilst also being inert for cells and biocompatible (Fig. 1 a, b). 29 , 30 Moreover, if combined with laser cutting, PVA is crucial for a seamless removal from the substrate, as otherwise the polymer fuses to the substrate at the cut edges. The coating functions as a separation, which is then dissolved, and the scaffold released, also offering the advantage of combining the washing steps and sterilization for cell culture if done in 70% ethanol. The issue of scaffold collapse, resulting from the weight of the liquid or surface tension upon wetting, causing deformation or entanglement, was addressed by employing FDM 3D printing reinforcements along the edges of the scaffolds (Fig. 1 a and b). These reinforcements maintained the scaffolds taut when wetted, providing a stable point for handling without direct contact with the scaffold itself. The polymers used for printing the reinforcements were PCL, PLA and PVA (Fig. 1 c, d and Extended Data Fig. 1 a), all FDA-approved, biocompatible and non-cytotoxic, rendering them ideal materials for cell culture applications. 31 – 33 The polymers adhered to the substrates without heating the 3D printer build plate, rendering compromises between 3D printer bed temperatures and scaffold integrity due to heating obsolete. Moreover, by adding glycerin to the PVA coating, the adhesion of the produced scaffold and FDM strut could be even further improved. Within this process, virtually any printable shapes and sizes of FDM reinforcements are possible. While we predominantly showcased single-layer circular reinforcements here, multi-layer high reinforcements are also applicable. Moreover, entire 3D structures, like components for bioreactors or inserts, can be directly printed onto the substrates and subsequently detached by dissolving the PVA base. 2.2. Investigation of potentially critical aspects of the new procedure Several experiments were conducted to critically assess potential issues in the new procedure. Firstly, the impact of temperatures during laser cutting and FDM printing on the delicate structure of the scaffolds was examined. Additionally, it was verified whether traces of the sacrificial layer made of PVA/Glycerin were detectable after detaching and washing the scaffolds. The mechanical resilience of the ring-reinforced fiber samples was evaluated through tensile tests and a practical bending test. Lastly, the question was addressed as to whether the method can be transferred to other materials. The results are presented below. Impact of temperature An important aspect to be considered in FDM printing on fiber constructs is the influence of heat during the extrusion of the melt. For instance, PLA and PVA have significantly higher melting points and, consequently, extrusion temperatures compared to the melt temperature of the PCL scaffolds produced here. To assess whether the delicate PCL structures are damaged by the radiant heat from the nozzle or the molten strand during the printing of support rings, a highly sensitive 20-layer PCL MEW scaffold with 4 µm fiber diameters was supported with PLA (printing temperature 180 ºC), PVA (printing temperature 190 ºC), and PCL (printing temperature 130 ºC). The transitions from the ring to the scaffold were then analyzed using SEM (Fig. 2 a). It was observed that the most significant influence occurs already during laser cutting, where the edges fuse due to heat radiation of the laser spot. Nevertheless, this is not a critical issue, as these regions will later be incorporated into the support ring. Reasonably less was seen on the edges of the PLA and PVA reinforced scaffolds, whilst with the PCL reinforcement, there was no trace thereof due to the lower printing temperature. The effects of excess heat damaging the scaffolds were generally minimal, only affecting the edges of the scaffolds if at all. Material variations To assess the general applicability of the method, several different scaffolds of various materials and characteristics were processed (Fig. 2 b and Extended Data Fig. 2 a). This consistently worked, except when combining PLA scaffolds with PCL reinforcement rings, as these tendentially separated from the scaffold, due to too low printing temperatures to bond the materials. Mechanical impact An important objective of the method is to make the samples resistant to mechanical influences during handling. To verify this, tensile tests and bending tests were conducted with various samples. As anticipated, the reinforcement samples exhibited distinct behavior under tensile stress (Fig. 2 c), with PCL being more flexible, while PLA and PVA were stiffer. Importantly, it was evident that the reinforced samples were highly resistant to deformation. Moreover, the reinforcement ring or the fiber scaffold would tear apart rather than separating from each other, as they were sufficiently fused (Extended Data Fig. 2 b). To simulate the practical scenario of transferring a wet sample, such as grasping it with tweezers, the samples underwent a semi-quantitative hanging test when dry and wetted. In this test, a straight scaffold would have a hanging angle of 0º, while a completely collapsed scaffold would measure an angle of 90º (Fig. 2 d). The dry scaffolds were straight and could support their own weight, whereas the unreinforced ones remained slightly misshapen (Fig. 2 e). Upon wetting, only the reinforced scaffolds maintained their hanging angle of 0º, while all others bent or completely collapsed, resulting in a change in angle. The varied hanging angles were influenced by factors such as fiber diameters, geometries, layer-to-layer adhesion, thickness, porosity, material, etc., indicating a high dependence on scaffold type for deformation and handleability. In contrast, the effects of inherent scaffold properties were nullified, and their stability significantly improved when reinforced with FDM. Residual sacrificial polymer The sacrificial PVA coating enables seamless detachment of the scaffolds form the substrates even in 70% ethanol. Thereafter the removal of the PVA residues from the scaffold is an important aspect, which was investigated using FTIR (Fig. 2 f and Extended Data Fig. 2 c), showing that after two washing steps any measurable traces of PVA were removed from electrowritten and electrospun scaffolds. 2.3. Application of the method to challenging scaffolds To demonstrate the versatile applicability of our procedure, we addressed several common challenges encountered when working with nanofiber-based materials: high-throughput scaffolds for quantitative screenings, super-fine scaffolds, and scaffolds for accurate placement. High throughput scaffolds Using a MEW scaffold, it was shown that, through automated laser cutting in combination with FDM printing, 137 precisely identical samples suitable for a 96-well plate (Fig. 3 c) could be processed within a short time (< 30 min) and with minimal effort. The material loss in this method is minimal, as almost the entire fiber mat can be utilized. Furthermore, an add-on method was developed to expedite the calibration step in FDM (Fig. 3 a) for complex sample geometries or frequently altered geometries. In this approach, a projector is employed, which can project onto the built plate of the printer using a mirror (Fig. 3 b and Extended Data Fig. 3 ). Initially, a calibration was printed onto the build plate, the projection aligned with it, and the print removed. Subsequently, the precise print location could be projected onto the laser-cut substrate when placed on the build plate. This feature makes it particularly suitable for multistep processing, as none of the preceding steps need to be calibrated or precise, thereby nullifying any cumulative errors that would otherwise be introduced by each processing step. Super fine scaffolds The utilization of advanced processing techniques allows for the handling of extremely thin scaffolds, exemplified by a one-layer box-shaped MEW PCL scaffold with a square edge length of 200 µm and a fiber diameter of 4 µm. This scaffold, suspended in the air and wetted at a 1 cm reinforcement ring diameter (Fig. 3 d), becomes feasible through the incorporation of reinforcement and PVA-facilitated removal. This approach extends to other thin and delicate scaffolds, provided that the scaffold material can adequately support its own weight and the surrounding liquid without undergoing deformation. Scaffolds for accurate placement In some applications, such as with implants, it may be necessary for a nanofiber construct to be precisely placed and sutured e.g. on native tissue. In such a case, it is essential to prevent the construct from collapsing or being damaged in a moist environment. While a reinforcing frame can prevent this, it may not be desired on the implant since it reduces its mechanical flexibility to adapt to organic topography and deformation. To demonstrate the suitability of the method for such applications, a reinforcing frame was 3D printed using PVA. It was shown that the dissolution of the PVA sacrificial coating to remove the scaffolds did not compromise the PVA reinforcement frame since it dissolves at a slower rate, and after detachment, further dissolution of the frame can be halted by immersion in isopropanol, an antisolvent (Fig. 3 e). PVA reinforcement may hence be used to stabilize the scaffold during handling and modification in non-aqueous media and maybe even assist whilst suturing scaffolds. This may be relevant for wound coverings, 35 – 37 heart patches or other implants, 38 , 39 where the biocompatible and low-cytotoxic frame could later dissolve in aqueous media (Fig. 3 f) or body fluids. Strikingly, the method could also be transferred to other selectively water-soluble polymers such as polyoxazolines or even tissue adhesive materials. 40 , 41 2.4 Two illustrative case studies: Standard cell culture and bioreactor application To demonstrate the added value of the new method, two case studies were conducted and documented. Firstly, highly sensitive samples were tested in a standard cell culture setting ( in-vitro test in well plates). In a second example, samples were prepared specifically for use in a bioreactor and subjected to treatment according to a representative protocol. The results of both case studies are presented below. Standard cell culture To assess the impact of FDM-printed reinforcements on cell culture outcomes, we compared ring-shaped scaffolds reinforced with PCL and PLA to their unreinforced counterparts. Initially, these scaffolds were evaluated alongside aligned electrospun PCL membranes using U87 murine glioblastoma cells, which are relatively insensitive. 42 – 44 Live/dead analysis revealed that the reinforcements did not significantly affect cell behavior, as cells performed similarly on all scaffolds (Fig. 4 a and Extended Data Fig. 4 a). The primary enhancement with the reinforcements was improved handleability, streamlining storage, removal, sterilization, and washing processes. The taut scaffold facilitated faster and easier manipulation, allowing for efficient transfer using tweezers. Since scaffold geometry influences cells, we assessed the impact of reinforcements on deformation-prone scaffolds utilizing thin MEW PCL scaffolds with triangular patterns. 45 These scaffolds were chosen due to their susceptibility to delamination and overall difficulty in handling, attributed to low layer-to-layer adhesion and thin fiber diameters. Murine Astrocytes, known for their sensitivity and preference for specific scaffold geometries like triangular grids, 46 , 47 were employed for this study to promote cell spreading conducive to proliferation. Live/dead results revealed significantly improved cell performance on FDM-reinforced scaffolds compared to unreinforced ones (Fig. 4 b and Extended Data Fig. 4 b). Closer examination (Fig. 4 c) using the inherently fluorescing PCL in the DAPI channel demonstrated that the form of the reinforced scaffolds was consistently maintained, remaining taut and significantly less prone to deformation. In contrast, the unreinforced scaffolds exhibited entanglement, collapse, and damage to pore size and geometry, likely hindering cell proliferation due to unfavorable conditions and potential cell entrapment. Moreover, these fluorescence images illustrated cells spreading across multiple fibers, emphasizing the risk of overstrain or tearing when deformation occurs in these fiber constructs. The reinforcements not only enhanced handleability but also exerted a significant impact on cell culture outcomes. Consequently, FDM reinforcements prove beneficial for handling across various scenarios, ensuring more consistent and reproducible results. By maintaining scaffolds in the desired shape, these reinforcements contribute to reducing artifacts, errors, and the production of unusable, unrepresentative scaffolds for evaluation post-experiment. Bioreactor application In applications like bioreactors, 47 , 48 spheroid culture, 49 or filtration, FDM-reinforced membranes outperform their unreinforced counterparts by maintaining tautness and uniformity when mounted into reactors (Fig. 5 a). Even large membranes, such as those made of a PCL/gelatin blend for lung models (Fig. 5 b), could be stabilized. 49 The variable shape of the reinforcement (Fig. 2 c) allows a customized fit, and soft materials like TPU A60 can serve as seals for bioreactors (Fig. 2 d, e, and f). To showcase enhanced handling and imaging capabilities, an FDM-reinforced electrospun random PCL membrane was incorporated into a bioreactor (Fig. 5 c-g). After a 3-day culture with Adipose-derived stem cells (ASCs), the membrane was extracted, fixed, and stained for actin and vinculin (Fig. 5 h). Subsequently, it was embedded in Mowiol for long-term storage and microscopy in a 3D printed chamber (Fig. 5 i1). Following imaging, the sample was withdrawn, suspended in a confocal dish in PBS, and imaged again for fibers and cells (Fig. 5 h and 5i2). The reinforcements prevented folding and deformation during dish movement, enhancing imaging quality. Post-imaging, any Mowiol residues were washed out, and the sample was prepared for SEM imaging using an ethanol drying procedure (Fig. 5 h and 5i3). The reinforcements facilitated seamless transfers and drying without surface contact, preventing sticking. Throughout the entire process, the integrity of both cells and the membrane, intentionally selected for its challenge in conventional handling, remained preserved. Cells on the membrane maintained a rounded shape, preventing the formation of focal adhesions observed in the treated glass control (Extended Data Fig. 5 ). Consequently, the FDM-reinforced membrane mitigated detachment issues commonly encountered with fragile fibers and weakly adhering cells, even after various handling and deformation steps (Fig. 5 i3). 3. Discussion Nano- and submicro-fibers hold immense significance across various applications, with thousands of annual publications dedicated solely to electrospinning. This study addresses the pressing need for scalable and standardized methods to improve accessibility to these promising materials for both laboratory research and industrial production. We have developed a scalable procedure using readily available and cost-effective technologies, enhancing consistency, production capacity, and time efficiency while also freeing up personnel resources. The new method involves sacrificial coating of the base substrate with PVA and reinforcing scaffold edges using FDM 3D printing with common biocompatible polymers. This combined approach facilitates easy detachment of scaffolds from the collector system, ensuring improved adhesion with the incorporation of glycerin in the PVA coating. Laser cutting and FDM 3D printing create scaffolds that are effortlessly removable and reinforced, maintaining tautness and preventing sample collapse or deformation when wetted during handling. Experimental assessments demonstrate notable improvements in cell culture applications, enhancing handleability, speed, and reliability of experiments, while preserving scaffold structure and shape. Furthermore, this system proves advantageous in bioreactors or filter-like applications, offering ease of application and post-processing with size and shape-tailorable FDM reinforcements. Soft plastics like TPU in FDM printing introduce bifunctional reinforcements, retaining scaffold shape and acting as a seal for reactor chambers. Moreover, the method allows for the design of geometrically, functionally, and physiologically customized membranes for personalized medicine, offering significant clinical benefits. The availability of standardized specimens in high throughput further simplifies the post-treatment and functionalization of the membranes, for example, as drug release systems, potentially increasing therapeutic intervention success and cost efficiency. By integrating biocompatible, water-soluble reinforcements, these systems have the potential to significantly impact diverse medical fields including dermatology, ophthalmology, neurodegenerative diseases, transplantation medicine, and immunology in the future. Their excellent applicability positions them as a promising bench-to-bedside strategy within the clinical realm. In summary, the method presented here enhances the availability of reproducible electrospun and electrowritten substrates in biofabrication and tissue engineering. It provides a cost-effective and easily implementable solution, resulting in significant improvements in cell culture and post-culture handling. This versatile approach allows for the customization of scaffolds to meet diverse requirements without sacrificing time efficiency or reproducibility, thereby fostering collaboration and standardization across laboratories. Thus, this work contributes to overcoming crucial bottlenecks in harnessing the potential of these fiber materials. 4. Methods PVA coating Polished steel plates or the rougher sides of aluminium foils, were cleaned with isopropanol, left to dry and then coated with a thin coat (ca. 0.03 ml/cm 2 ) of a 100 mg/ml solution of poly (vinyl alcohol) (PVA Mw 30–70 kDa, Merck KGaA, Darmstadt, Germany ) in water. The film was evenly distributed and left to dry at RT. When greater adhesion to the PVA film was desired, up to 11% (v/v) glycerin (Carl Roth, Karlsruhe, Germany) was added to the PVA solution before casting. Electrospinning of PCL membranes The PCL fiber membranes were produced by using a voltage difference of 9.5 kV, which was applied onto a 20 G needle (Microlance BD, New Jersey, USA). 1000 µl of the 24% w/v polycaprolactone (45 kDa, Sigma Aldrich, MO, USA) in 99% pure 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, abcr GmbH, Germany) solution were spun at a rate of 3 ml/h and a 17 cm distance between needle and collector. The grounded rotating drum collector (Ø 94 mm) was rotated at the speed of 1600 rpm for aligned and 100 rpm for random membranes. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30–70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector. The collected fiber membranes were submersed into a mixture of 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in H 2 O, dipped in 100% ethanol and dried. Electrospun PCL/gelatin membranes : 8% w/v PCL (Mw 80 kDa Merck KGaA, Darmstadt, Germany) and gelatin 2% w/v (type A from porcine skin, Merck KGaA, Darmstadt, Germany), were dissolved in a solvent mixture composed of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/formic acid (FA) (both from Sigma-Aldrich, HFIP:FA; 9:1 v/v). The solution was electrospun using a blunt 27G needle and the solution extruded at the speed of 0.3 ml/h, with a voltage of 15 kV applied to the needle. The grounded rotating drum collector (Ø 94 mm) was rotated at the speed of 100 rpm. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30–70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector. Electrospinning of PLA membranes A volume of 1.5 mL of a 2% Poly-L-Lactic Acid Mw 650 kDa (PLLA) (PL65 Purasorb, Netherlands) in HFIP solution was electrospun with a 27G nozzle, a voltage of 12 kV and a 15 cm distance from a rotating Ø 7 mm collector spinning at 100 rpm, to which, as previously mentioned, a PVA coated foil was attached. The collected fiber membranes were submersed into 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in 70% ethanol and dried. Production of PCL MEW scaffolds PCL box scaffolds were produced using melt electrowriting (MEW). The printing was conducted at a room temperature of 20 ºC with 40% humidity, a PCL (Purac PC12, Corbion, Amsterdam, the Netherlands) melt temperature of 95 ºC, a pressure of 1 bar and a print bed movement rate of 1000 mm/min onto a grounded steel build plate covered by a thin water-soluble poly (vinyl alcohol) (PVA, 30–70 kDa, Merck KGaA, Darmstadt, Germany) coating a 2.5 kV voltage difference applied over the 30 G needle (Nordson EFD, Ebensfeld, Germany), at a printing distance of 1.4 mm. Before application the PVA was dissolved using 70% ethanol, the scaffolds removed, washed in 70% ethanol and dried. The thicker electrowritten PCL scaffolds were produced using similar parameters, except that a 25G needle (Nordson EFD, Ebensfeld, Germany), a pressure of 2 bar and a speed of 500 mm/s were used. Laser cutting The scaffolds were laser cut (Rayjet, Trotec, Plymouth USA) to the appropriate well size. The speed and intensity of the laser were varied to achieve a complete separation of the remaining scaffold. FDM printing onto scaffolds The scaffolds were reinforced with polylactic acid (PLA, Form Futura, Amsterdam, Netherlands), polyvinyl alcohol (PVA) ( Form Futura, Amsterdam, Netherlands), or polycaprolactone (PCL) (Facilan Ortho, 3D4makers, Haarlem, The Netherlands) using a 0.4 mm nozzle, a layer height of 0.28 mm, a print speed of 5–20 mm/s, a 10 mm retraction distance and 80 mm/s retraction speed, without a heated print bed and the nozzle temperatures of 180 ⁰C, 190 ⁰C and 130 ⁰C for the different polymers respectively. Thermoplastic polyurethane (TPU) (FilaFlex 60A, Recreus, Elda, Spain) was printed using the same conditions, except that the nozzle temperature was 210 ºC and the retraction was disabled. Tensile testing The testing of the different reinforcement rings and scaffold was performed with a universal testing machine (Z010, Zwick Roell, Ulm, Germany) with a 100 N load cell. The samples were stretched with a velocity of 10 mm/min mounted between two clamps. The upper force limit was set to 95 N. The force dependent on the stretch was measured and evaluated. Hanging test wet/dry The hanging test was performed using a 3D printed construct into the beak of which the edges of the mesh were fastened. A background showing the different angles from 0° to 90° in steps of 10° was placed. The scaffolds were tested dry and wet, whereby the wet scaffolds were wetted by letting these absorb the liquid they could take up. A photograph was taken at a perpendicular height to the mesh. Triplicates were performed for each experiment and the angle of the hanging scaffold determined. Projector calibration To calibrate the projector (YABER V5, YABER, Austin, USA) to the build plate of the FDM printer (modified Ender 3 V2, Creality, Shenzen China), a calibration print was conducted, upon which the projection was calibrated. Thereafter the calibrated projection was used to calibrate the laser cut substrates to the correct position, where the printed strut would then be deposited. FTIR measurements The samples were measured dry using a Nicolet iS10 with smart iTR diamond ATR (attenuated total reflectance, Thermo Fisher Scientific, Waltham, USA). Scanning electron microscopy (SEM) The samples were analyzed using a SEM device (Crossbeam CB 340 SEM, Carl Zeiss). Ethanol drying procedure The already fixed cell samples were transferred to PBS and thoroughly washed. Afterwards these were incubated in 70%, 90% and 100% ethanol, twice for 10 min for each respective step. Thereafter the samples were incubated in hexamethyldisilazan (HMDS) (Merck KGaA, Darmstadt, Germany) twice for 10 min and subsequently left to dry. U87 culture and seeding (U-87 MG, ATCC HTB-14, LGC Standards GmbH, Germany) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (41966-029, Gibco, MA, USA) supplemented with 10% FCS (10270-106 Life Technologies, MA, USA) and 10,000 U/mL pen/strep (15140-122 Life Technologies, MA, USA). Cells were split twice per week. Scaffold were sterilized with 70% ethanol and were placed 15–30 min under UV light. Afterwards scaffolds were washed three times with ddH 2 O and once with PBS. 3 cm dishes with four 93 mm 2 inner rings (627170, Greiner, Greiner Bio-One, Kremsmünster, Austria) were used to place the scaffolds and add 50 µl of full media. Finally, scaffolds were incubated for 30 min at 37°C with 5% CO 2 , thereafter the cells were added at concentration of 10000 cells/well and further incubated. Ethical statement: Experiments were approved by the local veterinary authority (Veterinäramt der Stadt Würzburg, Germany) and the Ethics Committee of Animal Experiments, i.e., Regierung von Unterfranken, Würzburg, Germany (license no.: FBVVL 568/200-324/13). Astrocytes isolation and culture: CD-1 pups (P0-P1) were used to isolate primary astrocytes. After extracting the brains, cortices were dissected and collected in ice-cold phosphate-buffered saline (PBS). Following a brief homogenization and filtration through a 70 µm cell strainer (542070, Greiner Bio-One, Kremsmünster, Austria), cells were centrifuged (10 min, 1400 rpm), resuspended and seeded in 6 cm dishes with 5 mL of DMEM supplemented with 10% fetal calf serum, 2 × 10−3 m GlutaMAX, 1 × 10−3 m sodium pyruvate, and 50 U/mL penicillin/streptomycin (15140-122 Life Technologies, MA, USA). Astrocytes grew under standard conditions at 37 °C with 5% CO 2 . Cells were washed with PBS and medium was exchanged 3–4 days after seeding. After seven days, cells were detached and counted. 150,000 astrocytes in suspension were pipetted on top of each scaffold. An O-metal ring was used to fix the scaffolds. Afterward 3 mL of supplemented DMEM medium were added. Adipose-derived stem cell (ASCs) culture and seeding: Cells were centrifuged (5 min, 1200 rpm), resuspended and 15.000 cells were seeded on the electrospun membranes and on control glass slides in well plates with 5 mL of DMEM F-12 (1:1) supplemented with 200 mM GlutaMAX, 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA), 10% fetal calf serum, basic fibroblasts growth factor (FGF) and 50 µg/mL ascorbic acid (Sigma-Aldrich, Germany). ASC grew under standard conditions at 37 °C with 5% CO 2 atmosphere for three days. Medium was exchanged one day after seeding and every day after. Immunocytochemistry: For adipocytes the random electrospun PCL membranes were removed from the reactor by wetting the sides of the membrane outside of the main chamber with PBS Astrocyte/Adipocyte were washed once with PBS (pH 7.4) and fixed for 20 min with a 2% paraformaldehyde (PFA) solution or 3.7% gluteraldehyde. Following fixation, astrocytes were permeabilized and blocked with 5% normal goat serum (NGS) with 0.2 % Triton-X 100 in PBS for 30 min. Adipocytes were treated with 0.1% TritonX-100 in PBS for 5 minutes and blocked with 5% BSA in PBS for 30 min at room temperature. Astrocytes were incubated with ActinGreen™ 488 ReadyProbes™ Reagent (R37110 Invitrogen, Carlsbad, CA) in blocking solution for 1 hour. Finally, scaffolds were mounted with ProLong Glass Antifade Mountant containing Hoechst 33 342 (Thermo Fisher Scientific, Waltham, MA) on glass slides. Adipocytes were washed with PBS and incubated with primary antibody anti-vincullin (1:50; V4505 Sigma Aldrich, Germany) for 1 hour followed by secondary antibody incubation goat anti-rabbit-Cy3 (1:500, 111-165-003 Dianova, Hamburg, Germany). In the same step ActinGreen™ 488 readyProbes™ (1:50 R37110 Invitrogen, Carlsbad, CA) reagent staining was included. Cells were stained with DAPI (1:5000, D3571 Invitrogen, Canada) for 10 min and mounted on glass slides with Mowiol 4-88 (81381-50G Sigma Aldrich, Germany). The embedding was done in a FDM 3D printed chamber, slightly higher than the reinforced scaffold, and glued to a glass slide using nail lacquer, whilst the top was also sealed with glass and lacquer. This was done to easily embed the whole scaffolds and remove all bubbles. Live Dead staining of U87 cells and primary astrocytes: The staining was performed at day 1 and day 7 post-seeding at 21 °C for 20 min with Calcein-AM (2×10 −6 M, green/living cells; Thermo Fisher Scientific, Waltham, MA) and Ethidium Homodimer (2×10 −6 M, red/dead cells; Sigma-Aldrich, St. Louis, MO) diluted in PBS and incubated for 20 min. Confocal Microscopy and Image Acquisition: Samples were imaged using an inverted Olympus IX81 microscope equipped with an Olympus FV1000 confocal laser scanning system, a FVD10 SPD spectral detector, and diode lasers of 405 nm (DAPI), 473 nm (Alexa488) and 559 nm (Cy3) (Olympus, Tokyo, Japan). All images shown were acquired using an Olympus UPLSAPO 10× (air, numerical aperture 0.4) or Olympus UPLFLN 40x (oil, numerical aperture: 1.3) and were processed using ImageJ/Fiji 1 and Imaris 7.7.2 (Oxford Instrumentals, Abingdon, UK). For cell viability z-stacks of about 2–3.52 µm step size throughout each sample were acquired. Imaris was used for 3D reconstruction, video generation and reconstruction of the z-stack images to quantitatively analyze live and dead cell numbers The Spots function was used to determine the live/dead ratio. 5 image stacks per experimental condition were analyzed (N = 3). Dynamic range adjustments and projections were done with ImageJ/Fiji Software. 2.4. Statistical analysis GraphPad Prism 8.3.0 (Graphpad Software, San Diego, CA, USA) was used to calculate mean values, standard deviation (SD), standard error of the mean (SEM), and values for statistical significance. Statistical significance was estimated *p < 0.05 using two-way ANOVA. Declarations Acknowledgements This work was supported by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) under project number 326998133, within the framework of the Collaborative Research Center/Transregio 225 (SFB/TRR 225) "Biofabrication." The involved subprojects are A07 (PIs: Gregor Lang, Natascha Schäfer, and Dirk Schubert), C06 (PI: Taufiq Ahmad), and C05 (PI: Carmen Villmann). Additionally, we acknowledge funding from the DFG Priority Programme SPP 2416, CodeChi, under project number 525934737 (PIs: Sarah Zwingelberg and Gregor Lang). We extend our gratitude to the Graduate School of Life Sciences (GSLS) at the University of Würzburg for their support of our Ph.D. students. Additionally, the authors express their thanks to Dr. Thorsten Keller for providing the electrospun PCL/gelatin membrane and to Judith Friedlein for conducting SEM imaging. Received: ((will be filled in by the editorial staff)) Revised: ((will be filled in by the editorial staff)) Published online: ((will be filled in by the editorial staff)) References Gill, A. S., Sood, M., Deol, P. K. & Kaur, I. P. Synthetic polymer based electrospun scaffolds for wound healing applications. 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(\u003cstrong\u003eb\u003c/strong\u003e) electrospun fiber mats. (\u003cstrong\u003ec\u003c/strong\u003e) Melt-electrowritten scaffolds and (\u003cstrong\u003ed\u003c/strong\u003e) electrospun fiber mats after laser cutting, as well as with FDM printed support rings made of PLA, PVA and PCL. Scale bar for (\u003cstrong\u003ec\u003c/strong\u003e) and (\u003cstrong\u003ed\u003c/strong\u003e) corresponds to 5 mm.\u003c/p\u003e","description":"","filename":"floatimage1.png","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/0df7ee60212f98b2f0addf24.png"},{"id":53245842,"identity":"9df96bfc-8854-4ebf-b855-e0bb94a624ba","added_by":"auto","created_at":"2024-03-22 11:14:40","extension":"png","order_by":2,"title":"Figure 2","display":"","copyAsset":false,"role":"figure","size":594106,"visible":true,"origin":"","legend":"\u003cp\u003eDetailed analysis of reinforced scaffolds. (\u003cstrong\u003ea\u003c/strong\u003e) SEM images of electrowritten scaffolds after laser cutting, and reinforcement with PLA, PVA or PCL rings at the interface of the rings with the scaffolds. (\u003cstrong\u003eb\u003c/strong\u003e) Various other scaffolds made of either PCL or PLA reinforced with PLA rings. (\u003cstrong\u003ec\u003c/strong\u003e) Tensile tests of the different reinforcement rings and the thin MEW scaffold from \u003cstrong\u003ea\u003c/strong\u003e without rings. (\u003cstrong\u003ed\u003c/strong\u003e) Exemplary pictures of a semi-quantitative hanging angle test, conducted with dry and wet scaffolds, with and without reinforcement rings, showing the ability of the scaffolds to remain stable when dry and wetted, in this case using thin MEW scaffolds. (\u003cstrong\u003ee\u003c/strong\u003e) The hanging angle test results under wet conditions shown for scaffolds in \u003cstrong\u003ea\u003c/strong\u003e and \u003cstrong\u003ed\u003c/strong\u003e. (\u003cstrong\u003ef\u003c/strong\u003e) FTIR spectra showing the removal of the PVA from the MEW scaffolds after repeated washing steps in 70 % ethanol as indicated by the stepwise decrease of the PVA-characteristic broad O–H stretching band (3685–3010 cm\u003csup\u003e−1\u003c/sup\u003e)\u003csup\u003e34\u003c/sup\u003e. Scale bar (\u003cstrong\u003ea \u003c/strong\u003eand\u003cstrong\u003e b\u003c/strong\u003e) 400 µm and (\u003cstrong\u003ed\u003c/strong\u003e) 1 cm.\u003c/p\u003e","description":"","filename":"floatimage2.png","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/b1aeca17d2b6e920dfad84b6.png"},{"id":53245845,"identity":"6ea0a461-92ef-4d01-ad48-e338dd25e3ea","added_by":"auto","created_at":"2024-03-22 11:14:40","extension":"png","order_by":3,"title":"Figure 3","display":"","copyAsset":false,"role":"figure","size":1104538,"visible":true,"origin":"","legend":"\u003cp\u003eFurther improvements and capabilities of reinforced scaffolds. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic showing calibrating the printed pattern using a projection before FDM printing. (\u003cstrong\u003eb\u003c/strong\u003e) Projection calibration setup for the FDM printer, when using substrates that are not processed on the same device. (\u003cstrong\u003ec\u003c/strong\u003e) Image of a laser cut and reinforced scaffold with an appropriate size for 96 well plates, showing the precision that can be achieved using the projection calibration setup. (\u003cstrong\u003ed\u003c/strong\u003e) A wetted single layer MEW box scaffold suspended in the air, with the corresponding SEM images, showing the stability the reinforcement rings can provide even the most fragile unhandleable scaffolds. (\u003cstrong\u003ee\u003c/strong\u003e) A MEW scaffold with a PVA reinforcement ring after dissolution of the PVA base layer and subsequent immersion in isopropanol to stop further dissolution of the reinforcement. (\u003cstrong\u003ef\u003c/strong\u003e) A square PVA reinforced MEW scaffold being released from the reinforcement after dissolution of the ring in aqueous media. Scale bar (\u003cstrong\u003ec\u003c/strong\u003e) 5 mm, (\u003cstrong\u003ed\u003c/strong\u003e) 5 mm, 100 µm and 10 µm on magnifications, (\u003cstrong\u003ee\u003c/strong\u003e) 200 µm and (\u003cstrong\u003ef\u003c/strong\u003e) 1 cm.\u003c/p\u003e","description":"","filename":"floatimage3.png","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/a29f5e5729421102bf19445f.png"},{"id":53245844,"identity":"9c08ecb5-eef9-429b-9dfc-77f25d8e048e","added_by":"auto","created_at":"2024-03-22 11:14:40","extension":"png","order_by":4,"title":"Figure 4","display":"","copyAsset":false,"role":"figure","size":1241991,"visible":true,"origin":"","legend":"\u003cp\u003eCell cultural evaluation of reinforced and unreinforced scaffolds. (\u003cstrong\u003ea\u003c/strong\u003e) Live/dead on day 1 and 7 of U87 cells on aligned electrospun PCL scaffolds with and without PCL/PLA reinforcement rings with representative images of each condition on day 7. (\u003cstrong\u003eb\u003c/strong\u003e) Live/dead on day 1 and 7 of Astrocytes on MEW scaffolds unreinforced or reinforced with PCL/PLA rings, with representative images of day 7. (\u003cstrong\u003ec\u003c/strong\u003e) Images of Actin/DAPI staining depict form-stable reinforced scaffolds alongside misshapen unreinforced scaffolds, captured on both day 1 and day 7 under identical conditions as described in section B. The images also illustrate the scaffold geometry through DAPI staining. Plotted values in \u003cstrong\u003ea \u003c/strong\u003eand \u003cstrong\u003eb\u003c/strong\u003e presented as mean ± live/dead ratio, n = 3, three repetitions, p-values were calculated using a one-way ANOVA, *p \u0026lt; 0.05. Scale bar (\u003cstrong\u003ea\u003c/strong\u003e) 100 µm, (\u003cstrong\u003eb\u003c/strong\u003e) 200 µm and in (\u003cstrong\u003ec\u003c/strong\u003e) 50 µm.\u003c/p\u003e","description":"","filename":"floatimage4.png","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/2837c86ef199dfdec7ddf0dc.png"},{"id":53245841,"identity":"0360a8bd-0d3a-4748-a88d-5b9338423a0d","added_by":"auto","created_at":"2024-03-22 11:14:40","extension":"png","order_by":5,"title":"Figure 5","display":"","copyAsset":false,"role":"figure","size":669387,"visible":true,"origin":"","legend":"\u003cp\u003eImproved scaffolds in a complex application-oriented setting. (\u003cstrong\u003ea\u003c/strong\u003e) Schematic showing the difference of mounting a scaffold without or with reinforcement rings to a bioreactor or filtration setup. (\u003cstrong\u003eb\u003c/strong\u003e) A 7 cm diameter wetted electrospun PCL/gelatin membrane reinforced by a PLA ring, suspended in air. (\u003cstrong\u003ec\u003c/strong\u003e) A square random electrospun PCL membrane scaffold used for a bioreactor experiment, with a soft TPU reinforcement frame, which can also function as a seal. (\u003cstrong\u003ed\u003c/strong\u003e) The membrane from C, folded to show the TPU flexibility. (\u003cstrong\u003ee\u003c/strong\u003e) The membrane mounted onto one side of a two-chamber bioreactor. (\u003cstrong\u003ef\u003c/strong\u003e) The two assembled bioreactor halves. (\u003cstrong\u003eg\u003c/strong\u003e) The entire bioreactor setup mounted on a holder. (\u003cstrong\u003eh\u003c/strong\u003e) Illustration showing the processes endured by the membrane post-removal from the bioreactor after culturing. This involved primary/secondary anti-vinculin/actin antibody staining, embedding in a microscopy chamber in Mowiol for microscopy or long-term storage 1), subsequent washing out of the Mowiol and acquisition suspended in PBS using confocal microscopy to image the fibers and cells 2), followed by washing and the ethanol drying procedure for SEM imaging 3), all being conducted with the same sample. (\u003cstrong\u003ei\u003c/strong\u003e) The acquired images from the processing shown in \u003cstrong\u003eh\u003c/strong\u003e labeled with 1, 2 and 3. Scale bar (\u003cstrong\u003eb\u003c/strong\u003e) 2 cm, (\u003cstrong\u003ec\u003c/strong\u003e) 6 mm and in (\u003cstrong\u003ei\u003c/strong\u003e) 1-2) 50 µm 3) 80 µm and 20 µm on magnification.\u003c/p\u003e","description":"","filename":"floatimage5.png","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/d62050982f944d58427434ca.png"},{"id":71813351,"identity":"7de644dc-8082-4380-953d-9ac1439b730f","added_by":"auto","created_at":"2024-12-18 19:24:45","extension":"pdf","order_by":0,"title":"","display":"","copyAsset":false,"role":"manuscript-pdf","size":5531342,"visible":true,"origin":"","legend":"","description":"","filename":"manuscript.pdf","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/4ee94f2f-0792-4dcd-874e-abba145607e0.pdf"},{"id":53246289,"identity":"af3a0233-4771-451c-94c6-abe0a9ba401a","added_by":"auto","created_at":"2024-03-22 11:22:40","extension":"docx","order_by":1,"title":"","display":"","copyAsset":false,"role":"supplement","size":2652701,"visible":true,"origin":"","legend":"","description":"","filename":"Extendeddata.docx","url":"https://assets-eu.researchsquare.com/files/rs-4101827/v1/72cb76c2d602a58ad62cfbb5.docx"}],"financialInterests":"There is \u003cb\u003eNO\u003c/b\u003e Competing Interest.","formattedTitle":"Streamlining the highly reproducible fabrication of fibrous biomedical specimens towards standardization and high throughput","fulltext":[{"header":"1. Introduction","content":"\u003cp\u003eElectrostatic spinning methods, including electrospinning (ES) and melt electro-writing (MEW), have gained considerable attention in scientific research in recent decades. Their capability to create fiber diameters in the nano- and submicron ranges, along with the extensive diversity of synthetic and natural polymers available for use renders them particularly suitable for tissue engineering.\u003csup\u003e\u003cspan citationid=\"CR1\" class=\"CitationRef\"\u003e1\u003c/span\u003e,\u003cspan citationid=\"CR2\" class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e These methods facilitate the fabrication of biomimetic scaffolds resembling the fibrillar collagen structures present in natural tissues (50\u0026ndash;500 nm).\u003csup\u003e\u003cspan citationid=\"CR3\" class=\"CitationRef\"\u003e3\u003c/span\u003e\u003c/sup\u003e Furthermore, ongoing research investigates their potential in other medical applications such as wound coverings,\u003csup\u003e\u003cspan citationid=\"CR4\" class=\"CitationRef\"\u003e4\u003c/span\u003e\u003c/sup\u003e drug release,\u003csup\u003e\u003cspan citationid=\"CR5\" class=\"CitationRef\"\u003e5\u003c/span\u003e\u003c/sup\u003e as well as in the development of lab- and organ-on-a-chip devices.\u003csup\u003e\u003cspan citationid=\"CR6\" class=\"CitationRef\"\u003e6\u003c/span\u003e,\u003cspan citationid=\"CR7\" class=\"CitationRef\"\u003e7\u003c/span\u003e\u003c/sup\u003e Additionally, electrostatic spinning methods prove highly suitable for diverse technical applications due to the high surface-to-volume ratio of thin fibers and their remarkable porosity. These applications span various fields, including filtration,\u003csup\u003e\u003cspan citationid=\"CR8\" class=\"CitationRef\"\u003e8\u003c/span\u003e\u003c/sup\u003e textiles,\u003csup\u003e\u003cspan citationid=\"CR9\" class=\"CitationRef\"\u003e9\u003c/span\u003e\u003c/sup\u003e and sensing.\u003csup\u003e\u003cspan citationid=\"CR10\" class=\"CitationRef\"\u003e10\u003c/span\u003e,\u003cspan citationid=\"CR11\" class=\"CitationRef\"\u003e11\u003c/span\u003e\u003c/sup\u003e Despite their substantial potential, notably in the pharmaceutical sector, the commercial implementation of these material systems remains limited.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan citationid=\"CR13\" class=\"CitationRef\"\u003e13\u003c/span\u003e\u003c/sup\u003e However, it is anticipated that with advancing technological developments, their market for laboratory research and industrial production will witness substantial growth.\u003csup\u003e\u003cspan citationid=\"CR14\" class=\"CitationRef\"\u003e14\u003c/span\u003e\u003c/sup\u003e A recent review article by Ji et al. underscores the urgent need to devise innovative methods for scaling up nano-/micro-fiber materials while simultaneously preserving their quality and consistency.\u003csup\u003e\u003cspan citationid=\"CR15\" class=\"CitationRef\"\u003e15\u003c/span\u003e\u003c/sup\u003e Nadaf et al. also highlighted this crucial aspect, emphasizing that stability is the primary challenge when dealing with nanofibers.\u003csup\u003e\u003cspan citationid=\"CR16\" class=\"CitationRef\"\u003e16\u003c/span\u003e\u003c/sup\u003e Concurrently, there is a strong call to establish standards in processing and testing.\u003c/p\u003e \u003cp\u003eThe electrostatic spinning process itself is relatively simple, requiring only a high-voltage source, a needle pump, a collector, and a suitable polymer solution or melt. However, the processing of delicate fiber mats poses a significant challenge. Often, spinning occurs directly onto a grounded or oppositely charged collector plate, or for aligned fibers, onto a rotating cylinder. Subsequently, the fiber mats must be carefully peeled off for further preparation, a step that may lead to damage such as cracks, holes, or overstretching of the nonwoven membrane. In many applications, the fibers need to undergo chemical modifications or coatings for functionalization.\u003csup\u003e\u003cspan citationid=\"CR12\" class=\"CitationRef\"\u003e12\u003c/span\u003e,\u003cspan additionalcitationids=\"CR18 CR19 CR20\" citationid=\"CR17\" class=\"CitationRef\"\u003e17\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR21\" class=\"CitationRef\"\u003e21\u003c/span\u003e\u003c/sup\u003e This necessitates the transfer of samples into various agents, introducing mechanical stress and potential damage. With the advancement of increasingly precise analytical methods, such as those for characterizing the surfaces of functionalized fibers, the demands for the quality of the required samples also increase.\u003csup\u003e\u003cspan citationid=\"CR22\" class=\"CitationRef\"\u003e22\u003c/span\u003e\u003c/sup\u003e Correlative methods, for example, require the examination of a single sample in various devices, where selected spots must be precisely locatable. Wang et al. identified sample preparation as the greatest challenge in this regard and concluded, that especially in the field of biomaterials, novel fixation procedures need to be developed to better preserve cell and material structures.\u003csup\u003e\u003cspan citationid=\"CR23\" class=\"CitationRef\"\u003e23\u003c/span\u003e\u003c/sup\u003e In particular, thin nanofiber mats tend to collapse and entangle in a humid state, resulting in deformation and damage. Often, samples must be cut to an appropriate size for further testing or application, a process that can also compromise sensitive structures. In the past, individual solutions have been applied to address these challenges, such as detaching the nonwovens submersed in ethanol/water mixtures, low adhesive substrates or utilizing coatings such as gelatine.\u003csup\u003e\u003cspan additionalcitationids=\"CR25 CR26 CR27\" citationid=\"CR24\" class=\"CitationRef\"\u003e24\u003c/span\u003e\u0026ndash;\u003cspan citationid=\"CR28\" class=\"CitationRef\"\u003e28\u003c/span\u003e\u003c/sup\u003e Additionally, the fabrication of thicker fiber mats has been employed to enhance handling. Inserts have been used to immobilize samples, although this can lead to damage during the clamping of the nonwovens and, like the other strategies is hardly scalable. In summary, it can be affirmed that, at present, there is no universally applicable procedure to ensure the quality and integrity of nanofiber mats for various processing and application possibilities while consistently and adequately preparing samples in sufficient quantities. Frequently, the decision must be made between expedited handling that may cause damage to the scaffolds and laborious, slow work aimed at preserving the structures. This choice has significant implications for the reproducibility of results.\u003c/p\u003e \u003cp\u003eTo address this issue, we developed a new workflow that is not only easily implementable, universally applicable, scalable, and cost-effective but also can be carried out with standard laboratory equipment or minimal investment costs. Applying a sacrificial film as a substrate material in combination with laser cutting and fused deposition modeling (FDM) resulted in high throughput production of electrospun and melt-electrowritten scaffolds, that are perfectly manageable for virtually any subsequent step, be it analytical or application-oriented.\u003c/p\u003e"},{"header":"2. Results","content":"\u003cdiv id=\"Sec3\" class=\"Section2\"\u003e\n\u003ch2\u003e2.1. Basic workflow to harvest electrospun and electrowritten scaffolds\u003c/h2\u003e\n\u003cp\u003eTo facilitate seamless removal of the electrospun or electrowritten scaffolds from their substrates after production, we used a water/ethanol soluble sacrificial cast PVA coating cast on steel plates or aluminium foil. The FDA-approved PVA is known to not adsorb to polymers that constitute the scaffolds, whilst also being inert for cells and biocompatible (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea, b).\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e29\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e30\u003c/span\u003e\u003c/sup\u003e Moreover, if combined with laser cutting, PVA is crucial for a seamless removal from the substrate, as otherwise the polymer fuses to the substrate at the cut edges. The coating functions as a separation, which is then dissolved, and the scaffold released, also offering the advantage of combining the washing steps and sterilization for cell culture if done in 70% ethanol.\u003c/p\u003e\n\u003cp\u003eThe issue of scaffold collapse, resulting from the weight of the liquid or surface tension upon wetting, causing deformation or entanglement, was addressed by employing FDM 3D printing reinforcements along the edges of the scaffolds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea and b). These reinforcements maintained the scaffolds taut when wetted, providing a stable point for handling without direct contact with the scaffold itself. The polymers used for printing the reinforcements were PCL, PLA and PVA (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ec, d and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e1\u003c/span\u003ea), all FDA-approved, biocompatible and non-cytotoxic, rendering them ideal materials for cell culture applications.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e31\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e33\u003c/span\u003e\u003c/sup\u003e The polymers adhered to the substrates without heating the 3D printer build plate, rendering compromises between 3D printer bed temperatures and scaffold integrity due to heating obsolete. Moreover, by adding glycerin to the PVA coating, the adhesion of the produced scaffold and FDM strut could be even further improved. Within this process, virtually any printable shapes and sizes of FDM reinforcements are possible. While we predominantly showcased single-layer circular reinforcements here, multi-layer high reinforcements are also applicable. Moreover, entire 3D structures, like components for bioreactors or inserts, can be directly printed onto the substrates and subsequently detached by dissolving the PVA base.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec4\" class=\"Section2\"\u003e\n\u003ch2\u003e2.2. Investigation of potentially critical aspects of the new procedure\u003c/h2\u003e\n\u003cp\u003eSeveral experiments were conducted to critically assess potential issues in the new procedure. Firstly, the impact of temperatures during laser cutting and FDM printing on the delicate structure of the scaffolds was examined. Additionally, it was verified whether traces of the sacrificial layer made of PVA/Glycerin were detectable after detaching and washing the scaffolds. The mechanical resilience of the ring-reinforced fiber samples was evaluated through tensile tests and a practical bending test. Lastly, the question was addressed as to whether the method can be transferred to other materials. The results are presented below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eImpact of temperature\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn important aspect to be considered in FDM printing on fiber constructs is the influence of heat during the extrusion of the melt. For instance, PLA and PVA have significantly higher melting points and, consequently, extrusion temperatures compared to the melt temperature of the PCL scaffolds produced here. To assess whether the delicate PCL structures are damaged by the radiant heat from the nozzle or the molten strand during the printing of support rings, a highly sensitive 20-layer PCL MEW scaffold with 4 \u0026micro;m fiber diameters was supported with PLA (printing temperature 180 \u0026ordm;C), PVA (printing temperature 190 \u0026ordm;C), and PCL (printing temperature 130 \u0026ordm;C). The transitions from the ring to the scaffold were then analyzed using SEM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). It was observed that the most significant influence occurs already during laser cutting, where the edges fuse due to heat radiation of the laser spot. Nevertheless, this is not a critical issue, as these regions will later be incorporated into the support ring. Reasonably less was seen on the edges of the PLA and PVA reinforced scaffolds, whilst with the PCL reinforcement, there was no trace thereof due to the lower printing temperature. The effects of excess heat damaging the scaffolds were generally minimal, only affecting the edges of the scaffolds if at all.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMaterial variations\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the general applicability of the method, several different scaffolds of various materials and characteristics were processed (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ea). This consistently worked, except when combining PLA scaffolds with PCL reinforcement rings, as these tendentially separated from the scaffold, due to too low printing temperatures to bond the materials.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eMechanical impact\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eAn important objective of the method is to make the samples resistant to mechanical influences during handling. To verify this, tensile tests and bending tests were conducted with various samples. As anticipated, the reinforcement samples exhibited distinct behavior under tensile stress (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), with PCL being more flexible, while PLA and PVA were stiffer. Importantly, it was evident that the reinforced samples were highly resistant to deformation. Moreover, the reinforcement ring or the fiber scaffold would tear apart rather than separating from each other, as they were sufficiently fused (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003eb). To simulate the practical scenario of transferring a wet sample, such as grasping it with tweezers, the samples underwent a semi-quantitative hanging test when dry and wetted. In this test, a straight scaffold would have a hanging angle of 0\u0026ordm;, while a completely collapsed scaffold would measure an angle of 90\u0026ordm; (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed). The dry scaffolds were straight and could support their own weight, whereas the unreinforced ones remained slightly misshapen (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ee). Upon wetting, only the reinforced scaffolds maintained their hanging angle of 0\u0026ordm;, while all others bent or completely collapsed, resulting in a change in angle. The varied hanging angles were influenced by factors such as fiber diameters, geometries, layer-to-layer adhesion, thickness, porosity, material, etc., indicating a high dependence on scaffold type for deformation and handleability. In contrast, the effects of inherent scaffold properties were nullified, and their stability significantly improved when reinforced with FDM.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eResidual sacrificial polymer\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe sacrificial PVA coating enables seamless detachment of the scaffolds form the substrates even in 70% ethanol. Thereafter the removal of the PVA residues from the scaffold is an important aspect, which was investigated using FTIR (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ef and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec), showing that after two washing steps any measurable traces of PVA were removed from electrowritten and electrospun scaffolds.\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec5\" class=\"Section2\"\u003e\n\u003ch2\u003e2.3. Application of the method to challenging scaffolds\u003c/h2\u003e\n\u003cp\u003eTo demonstrate the versatile applicability of our procedure, we addressed several common challenges encountered when working with nanofiber-based materials: high-throughput scaffolds for quantitative screenings, super-fine scaffolds, and scaffolds for accurate placement.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHigh throughput scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eUsing a MEW scaffold, it was shown that, through automated laser cutting in combination with FDM printing, 137 precisely identical samples suitable for a 96-well plate (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ec) could be processed within a short time (\u0026lt;\u0026thinsp;30 min) and with minimal effort. The material loss in this method is minimal, as almost the entire fiber mat can be utilized. Furthermore, an add-on method was developed to expedite the calibration step in FDM (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ea) for complex sample geometries or frequently altered geometries. In this approach, a projector is employed, which can project onto the built plate of the printer using a mirror (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003e). Initially, a calibration was printed onto the build plate, the projection aligned with it, and the print removed. Subsequently, the precise print location could be projected onto the laser-cut substrate when placed on the build plate. This feature makes it particularly suitable for multistep processing, as none of the preceding steps need to be calibrated or precise, thereby nullifying any cumulative errors that would otherwise be introduced by each processing step.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eSuper fine scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe utilization of advanced processing techniques allows for the handling of extremely thin scaffolds, exemplified by a one-layer box-shaped MEW PCL scaffold with a square edge length of 200 \u0026micro;m and a fiber diameter of 4 \u0026micro;m. This scaffold, suspended in the air and wetted at a 1 cm reinforcement ring diameter (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ed), becomes feasible through the incorporation of reinforcement and PVA-facilitated removal. This approach extends to other thin and delicate scaffolds, provided that the scaffold material can adequately support its own weight and the surrounding liquid without undergoing deformation.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScaffolds for accurate placement\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn some applications, such as with implants, it may be necessary for a nanofiber construct to be precisely placed and sutured e.g. on native tissue. In such a case, it is essential to prevent the construct from collapsing or being damaged in a moist environment. While a reinforcing frame can prevent this, it may not be desired on the implant since it reduces its mechanical flexibility to adapt to organic topography and deformation. To demonstrate the suitability of the method for such applications, a reinforcing frame was 3D printed using PVA. It was shown that the dissolution of the PVA sacrificial coating to remove the scaffolds did not compromise the PVA reinforcement frame since it dissolves at a slower rate, and after detachment, further dissolution of the frame can be halted by immersion in isopropanol, an antisolvent (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ee). PVA reinforcement may hence be used to stabilize the scaffold during handling and modification in non-aqueous media and maybe even assist whilst suturing scaffolds. This may be relevant for wound coverings,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e35\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e37\u003c/span\u003e\u003c/sup\u003e heart patches or other implants,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e38\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e39\u003c/span\u003e\u003c/sup\u003e where the biocompatible and low-cytotoxic frame could later dissolve in aqueous media (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e3\u003c/span\u003ef) or body fluids. Strikingly, the method could also be transferred to other selectively water-soluble polymers such as polyoxazolines or even tissue adhesive materials.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e40\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e41\u003c/span\u003e\u003c/sup\u003e\u003c/p\u003e\n\u003c/div\u003e\n\u003cdiv id=\"Sec6\" class=\"Section2\"\u003e\n\u003ch2\u003e2.4 Two illustrative case studies: Standard cell culture and bioreactor application\u003c/h2\u003e\n\u003cp\u003eTo demonstrate the added value of the new method, two case studies were conducted and documented. Firstly, highly sensitive samples were tested in a standard cell culture setting (\u003cem\u003ein-vitro\u003c/em\u003e test in well plates). In a second example, samples were prepared specifically for use in a bioreactor and subjected to treatment according to a representative protocol. The results of both case studies are presented below.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eStandard cell culture\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo assess the impact of FDM-printed reinforcements on cell culture outcomes, we compared ring-shaped scaffolds reinforced with PCL and PLA to their unreinforced counterparts. Initially, these scaffolds were evaluated alongside aligned electrospun PCL membranes using U87 murine glioblastoma cells, which are relatively insensitive.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e42\u003c/span\u003e\u0026ndash;\u003cspan class=\"CitationRef\"\u003e44\u003c/span\u003e\u003c/sup\u003e Live/dead analysis revealed that the reinforcements did not significantly affect cell behavior, as cells performed similarly on all scaffolds (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ea). The primary enhancement with the reinforcements was improved handleability, streamlining storage, removal, sterilization, and washing processes. The taut scaffold facilitated faster and easier manipulation, allowing for efficient transfer using tweezers.\u003c/p\u003e\n\u003cp\u003eSince scaffold geometry influences cells, we assessed the impact of reinforcements on deformation-prone scaffolds utilizing thin MEW PCL scaffolds with triangular patterns.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e45\u003c/span\u003e\u003c/sup\u003e These scaffolds were chosen due to their susceptibility to delamination and overall difficulty in handling, attributed to low layer-to-layer adhesion and thin fiber diameters. Murine Astrocytes, known for their sensitivity and preference for specific scaffold geometries like triangular grids,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e46\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e\u003c/sup\u003e were employed for this study to promote cell spreading conducive to proliferation. Live/dead results revealed significantly improved cell performance on FDM-reinforced scaffolds compared to unreinforced ones (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb and Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003eb). Closer examination (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e4\u003c/span\u003ec) using the inherently fluorescing PCL in the DAPI channel demonstrated that the form of the reinforced scaffolds was consistently maintained, remaining taut and significantly less prone to deformation. In contrast, the unreinforced scaffolds exhibited entanglement, collapse, and damage to pore size and geometry, likely hindering cell proliferation due to unfavorable conditions and potential cell entrapment. Moreover, these fluorescence images illustrated cells spreading across multiple fibers, emphasizing the risk of overstrain or tearing when deformation occurs in these fiber constructs.\u003c/p\u003e\n\u003cp\u003eThe reinforcements not only enhanced handleability but also exerted a significant impact on cell culture outcomes. Consequently, FDM reinforcements prove beneficial for handling across various scenarios, ensuring more consistent and reproducible results. By maintaining scaffolds in the desired shape, these reinforcements contribute to reducing artifacts, errors, and the production of unusable, unrepresentative scaffolds for evaluation post-experiment.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eBioreactor application\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eIn applications like bioreactors,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e47\u003c/span\u003e,\u003cspan class=\"CitationRef\"\u003e48\u003c/span\u003e\u003c/sup\u003e spheroid culture,\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e or filtration, FDM-reinforced membranes outperform their unreinforced counterparts by maintaining tautness and uniformity when mounted into reactors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ea). Even large membranes, such as those made of a PCL/gelatin blend for lung models (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eb), could be stabilized.\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e49\u003c/span\u003e\u003c/sup\u003e The variable shape of the reinforcement (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ec) allows a customized fit, and soft materials like TPU A60 can serve as seals for bioreactors (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e2\u003c/span\u003ed, e, and f).\u003c/p\u003e\n\u003cp\u003eTo showcase enhanced handling and imaging capabilities, an FDM-reinforced electrospun random PCL membrane was incorporated into a bioreactor (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ec-g). After a 3-day culture with Adipose-derived stem cells (ASCs), the membrane was extracted, fixed, and stained for actin and vinculin (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh). Subsequently, it was embedded in Mowiol for long-term storage and microscopy in a 3D printed chamber (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ei1). Following imaging, the sample was withdrawn, suspended in a confocal dish in PBS, and imaged again for fibers and cells (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh and 5i2). The reinforcements prevented folding and deformation during dish movement, enhancing imaging quality. Post-imaging, any Mowiol residues were washed out, and the sample was prepared for SEM imaging using an ethanol drying procedure (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003eh and 5i3). The reinforcements facilitated seamless transfers and drying without surface contact, preventing sticking. Throughout the entire process, the integrity of both cells and the membrane, intentionally selected for its challenge in conventional handling, remained preserved. Cells on the membrane maintained a rounded shape, preventing the formation of focal adhesions observed in the treated glass control (Extended Data Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003e). Consequently, the FDM-reinforced membrane mitigated detachment issues commonly encountered with fragile fibers and weakly adhering cells, even after various handling and deformation steps (Fig.\u0026nbsp;\u003cspan class=\"InternalRef\"\u003e5\u003c/span\u003ei3).\u003c/p\u003e\n\u003c/div\u003e"},{"header":"3. Discussion","content":"\u003cp\u003eNano- and submicro-fibers hold immense significance across various applications, with thousands of annual publications dedicated solely to electrospinning. This study addresses the pressing need for scalable and standardized methods to improve accessibility to these promising materials for both laboratory research and industrial production. We have developed a scalable procedure using readily available and cost-effective technologies, enhancing consistency, production capacity, and time efficiency while also freeing up personnel resources. The new method involves sacrificial coating of the base substrate with PVA and reinforcing scaffold edges using FDM 3D printing with common biocompatible polymers. This combined approach facilitates easy detachment of scaffolds from the collector system, ensuring improved adhesion with the incorporation of glycerin in the PVA coating. Laser cutting and FDM 3D printing create scaffolds that are effortlessly removable and reinforced, maintaining tautness and preventing sample collapse or deformation when wetted during handling. Experimental assessments demonstrate notable improvements in cell culture applications, enhancing handleability, speed, and reliability of experiments, while preserving scaffold structure and shape.\u003c/p\u003e \u003cp\u003eFurthermore, this system proves advantageous in bioreactors or filter-like applications, offering ease of application and post-processing with size and shape-tailorable FDM reinforcements. Soft plastics like TPU in FDM printing introduce bifunctional reinforcements, retaining scaffold shape and acting as a seal for reactor chambers. Moreover, the method allows for the design of geometrically, functionally, and physiologically customized membranes for personalized medicine, offering significant clinical benefits. The availability of standardized specimens in high throughput further simplifies the post-treatment and functionalization of the membranes, for example, as drug release systems, potentially increasing therapeutic intervention success and cost efficiency. By integrating biocompatible, water-soluble reinforcements, these systems have the potential to significantly impact diverse medical fields including dermatology, ophthalmology, neurodegenerative diseases, transplantation medicine, and immunology in the future. Their excellent applicability positions them as a promising bench-to-bedside strategy within the clinical realm.\u003c/p\u003e \u003cp\u003eIn summary, the method presented here enhances the availability of reproducible electrospun and electrowritten substrates in biofabrication and tissue engineering. It provides a cost-effective and easily implementable solution, resulting in significant improvements in cell culture and post-culture handling. This versatile approach allows for the customization of scaffolds to meet diverse requirements without sacrificing time efficiency or reproducibility, thereby fostering collaboration and standardization across laboratories. Thus, this work contributes to overcoming crucial bottlenecks in harnessing the potential of these fiber materials.\u003c/p\u003e"},{"header":"4. Methods","content":"\u003cp\u003e\u003cstrong\u003ePVA coating\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePolished steel plates or the rougher sides of aluminium foils, were cleaned with isopropanol, left to dry and then coated with a thin coat (ca. 0.03 ml/cm\u003csup\u003e2\u003c/sup\u003e) of a 100 mg/ml solution of poly (vinyl alcohol) (PVA Mw 30\u0026ndash;70 kDa, Merck KGaA, Darmstadt, Germany\u003cem\u003e)\u003c/em\u003e in water. The film was evenly distributed and left to dry at RT. When greater adhesion to the PVA film was desired, up to 11% (v/v) glycerin (Carl Roth, Karlsruhe, Germany) was added to the PVA solution before casting.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrospinning of PCL membranes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe PCL fiber membranes were produced by using a voltage difference of 9.5 kV, which was applied onto a 20 G needle (Microlance BD, New Jersey, USA). 1000 \u0026micro;l of the 24% w/v polycaprolactone (45 kDa, Sigma Aldrich, MO, USA) in 99% pure 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP, abcr GmbH, Germany) solution were spun at a rate of 3 ml/h and a 17 cm distance between needle and collector. The grounded rotating drum collector (\u0026Oslash; 94 mm) was rotated at the speed of 1600 rpm for aligned and 100 rpm for random membranes. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30\u0026ndash;70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector. The collected fiber membranes were submersed into a mixture of 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in H\u003csub\u003e2\u003c/sub\u003eO, dipped in 100% ethanol and dried.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eElectrospun PCL/gelatin membranes\u003c/em\u003e: 8% w/v PCL (Mw 80 kDa Merck KGaA, Darmstadt, Germany) and gelatin 2% w/v (type A from porcine skin, Merck KGaA, Darmstadt, Germany), were dissolved in a solvent mixture composed of 1,1,1,3,3,3-hexafluoro-2-propanol (HFIP)/formic acid (FA) (both from Sigma-Aldrich, HFIP:FA; 9:1 v/v). The solution was electrospun using a blunt 27G needle and the solution extruded at the speed of 0.3 ml/h, with a voltage of 15 kV applied to the needle. The grounded rotating drum collector (\u0026Oslash; 94 mm) was rotated at the speed of 100 rpm. The membranes were spun onto an aluminium foil thinly coated with polyvinyl alcohol (PVA 30\u0026ndash;70 kDa, Merck KGaA, Darmstadt, Germany) that was attached to the collector.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eElectrospinning of PLA membranes\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eA volume of 1.5 mL of a 2% Poly-L-Lactic Acid Mw 650 kDa (PLLA) (PL65 Purasorb, Netherlands) in HFIP solution was electrospun with a 27G nozzle, a voltage of 12 kV and a 15 cm distance from a rotating \u0026Oslash; 7 mm collector spinning at 100 rpm, to which, as previously mentioned, a PVA coated foil was attached. The collected fiber membranes were submersed into 70% ethanol (v/v) and soaked for ca. 1 min. The membranes were then washed thrice in 70% ethanol and dried.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProduction of PCL MEW scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003ePCL box scaffolds were produced using melt electrowriting (MEW). The printing was conducted at a room temperature of 20 \u0026ordm;C with 40% humidity, a PCL (Purac PC12, Corbion, Amsterdam, the Netherlands) melt temperature of 95 \u0026ordm;C, a pressure of 1 bar and a print bed movement rate of 1000 mm/min onto a grounded steel build plate covered by a thin water-soluble poly (vinyl alcohol) (PVA, 30\u0026ndash;70 kDa, Merck KGaA, Darmstadt, Germany) coating a 2.5 kV voltage difference applied over the 30 G needle (Nordson EFD, Ebensfeld, Germany), at a printing distance of 1.4 mm. Before application the PVA was dissolved using 70% ethanol, the scaffolds removed, washed in 70% ethanol and dried.\u003c/p\u003e\n\u003cp\u003eThe thicker electrowritten PCL scaffolds were produced using similar parameters, except that a 25G needle (Nordson EFD, Ebensfeld, Germany), a pressure of 2 bar and a speed of 500 mm/s were used.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eLaser cutting\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scaffolds were laser cut (Rayjet, Trotec, Plymouth USA) to the appropriate well size. The speed and intensity of the laser were varied to achieve a complete separation of the remaining scaffold.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFDM printing onto scaffolds\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe scaffolds were reinforced with polylactic acid (PLA, Form Futura, Amsterdam, Netherlands), polyvinyl alcohol (PVA) ( Form Futura, Amsterdam, Netherlands), or polycaprolactone (PCL) (Facilan Ortho, 3D4makers, Haarlem, The Netherlands) using a 0.4 mm nozzle, a layer height of 0.28 mm, a print speed of 5\u0026ndash;20 mm/s, a 10 mm retraction distance and 80 mm/s retraction speed, without a heated print bed and the nozzle temperatures of 180 ⁰C, 190 ⁰C and 130 ⁰C for the different polymers respectively. Thermoplastic polyurethane (TPU) (FilaFlex 60A, Recreus, Elda, Spain) was printed using the same conditions, except that the nozzle temperature was 210 \u0026ordm;C and the retraction was disabled.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eTensile testing\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe testing of the different reinforcement rings and scaffold was performed with a universal testing machine (Z010, Zwick Roell, Ulm, Germany) with a 100 N load cell. The samples were stretched with a velocity of 10 mm/min mounted between two clamps. The upper force limit was set to 95 N. The force dependent on the stretch was measured and evaluated.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eHanging test wet/dry\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe hanging test was performed using a 3D printed construct into the beak of which the edges of the mesh were fastened. A background showing the different angles from 0\u0026deg; to 90\u0026deg; in steps of 10\u0026deg; was placed. The scaffolds were tested dry and wet, whereby the wet scaffolds were wetted by letting these absorb the liquid they could take up. A photograph was taken at a perpendicular height to the mesh. Triplicates were performed for each experiment and the angle of the hanging scaffold determined.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eProjector calibration\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eTo calibrate the projector (YABER V5, YABER, Austin, USA) to the build plate of the FDM printer (modified Ender 3 V2, Creality, Shenzen China), a calibration print was conducted, upon which the projection was calibrated. Thereafter the calibrated projection was used to calibrate the laser cut substrates to the correct position, where the printed strut would then be deposited.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eFTIR measurements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were measured dry using a Nicolet iS10 with smart iTR diamond ATR (attenuated total reflectance, Thermo Fisher Scientific, Waltham, USA).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eScanning electron microscopy (SEM)\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe samples were analyzed using a SEM device (Crossbeam CB 340 SEM, Carl Zeiss).\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eEthanol drying procedure\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThe already fixed cell samples were transferred to PBS and thoroughly washed. Afterwards these were incubated in 70%, 90% and 100% ethanol, twice for 10 min for each respective step. Thereafter the samples were incubated in hexamethyldisilazan (HMDS) (Merck KGaA, Darmstadt, Germany) twice for 10 min and subsequently left to dry.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003eU87 culture and seeding\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003e(U-87 MG, ATCC HTB-14, LGC Standards GmbH, Germany) were cultured in Dulbecco's Modified Eagle Medium (DMEM) (41966-029, Gibco, MA, USA) supplemented with 10% FCS (10270-106 Life Technologies, MA, USA) and 10,000 U/mL pen/strep (15140-122 Life Technologies, MA, USA). Cells were split twice per week.\u003c/p\u003e\n\u003cp\u003eScaffold were sterilized with 70% ethanol and were placed 15\u0026ndash;30 min under UV light. Afterwards scaffolds were washed three times with ddH\u003csub\u003e2\u003c/sub\u003eO and once with PBS. 3 cm dishes with four 93 mm\u003csup\u003e\u003cspan class=\"CitationRef\"\u003e2\u003c/span\u003e\u003c/sup\u003e inner rings (627170, Greiner, Greiner Bio-One, Kremsm\u0026uuml;nster, Austria) were used to place the scaffolds and add 50 \u0026micro;l of full media. Finally, scaffolds were incubated for 30 min at 37\u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e, thereafter the cells were added at concentration of 10000 cells/well and further incubated.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eEthical statement:\u0026nbsp;\u003c/em\u003eExperiments were approved by the local veterinary authority (Veterin\u0026auml;ramt der Stadt W\u0026uuml;rzburg, Germany) and the Ethics Committee of Animal Experiments, i.e., Regierung von Unterfranken, W\u0026uuml;rzburg, Germany (license no.: FBVVL 568/200-324/13).\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAstrocytes isolation and culture:\u0026nbsp;\u003c/em\u003eCD-1 pups (P0-P1) were used to isolate primary astrocytes. After extracting the brains, cortices were dissected and collected in ice-cold phosphate-buffered saline (PBS). Following a brief homogenization and filtration through a 70 \u0026micro;m cell strainer (542070, Greiner Bio-One, Kremsm\u0026uuml;nster, Austria), cells were centrifuged (10 min, 1400 rpm), resuspended and seeded in 6 cm dishes with 5 mL of DMEM supplemented with 10% fetal calf serum, 2 \u0026times; 10\u0026minus;3 m GlutaMAX, 1 \u0026times; 10\u0026minus;3 m sodium pyruvate, and 50 U/mL penicillin/streptomycin (15140-122 Life Technologies, MA, USA). Astrocytes grew under standard conditions at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e. Cells were washed with PBS and medium was exchanged 3\u0026ndash;4 days after seeding. After seven days, cells were detached and counted. 150,000 astrocytes in suspension were pipetted on top of each scaffold. An O-metal ring was used to fix the scaffolds. Afterward 3 mL of supplemented DMEM medium were added.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eAdipose-derived stem cell (ASCs)\u0026nbsp;\u003c/em\u003e\u003cem\u003eculture and seeding:\u003c/em\u003e Cells were centrifuged (5 min, 1200 rpm), resuspended and 15.000 cells were seeded on the electrospun membranes and on control glass slides in well plates with 5 mL of DMEM F-12 (1:1) supplemented with 200 mM GlutaMAX, 100 U/mL penicillin/streptomycin (Thermo Fisher Scientific, Waltham, MA), 10% fetal calf serum, basic fibroblasts growth factor (FGF) and 50 \u0026micro;g/mL ascorbic acid (Sigma-Aldrich, Germany). ASC grew under standard conditions at 37 \u0026deg;C with 5% CO\u003csub\u003e2\u003c/sub\u003e atmosphere for three days. Medium was exchanged one day after seeding and every day after.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eImmunocytochemistry:\u003c/em\u003e For adipocytes the random electrospun PCL membranes were removed from the reactor by wetting the sides of the membrane outside of the main chamber with PBS Astrocyte/Adipocyte were washed once with PBS (pH 7.4) and fixed for 20 min with a 2% paraformaldehyde (PFA) solution or 3.7% gluteraldehyde. Following fixation, astrocytes were permeabilized and blocked with 5% normal goat serum (NGS) with 0.2 % Triton-X 100 in PBS for 30 min. Adipocytes were treated with 0.1% TritonX-100 in PBS for 5 minutes and blocked with 5% BSA in PBS for 30 min at room temperature. Astrocytes were incubated with ActinGreen\u0026trade; 488 ReadyProbes\u0026trade; Reagent (R37110 Invitrogen, Carlsbad, CA) in blocking solution for 1 hour. Finally, scaffolds were mounted with ProLong Glass Antifade Mountant containing Hoechst 33 342 (Thermo Fisher Scientific, Waltham, MA) on glass slides. Adipocytes were washed with PBS and incubated with primary antibody anti-vincullin (1:50; V4505 Sigma Aldrich, Germany) for 1 hour followed by secondary antibody incubation goat anti-rabbit-Cy3 (1:500, 111-165-003 Dianova, Hamburg, Germany). In the same step ActinGreen\u0026trade; 488 readyProbes\u0026trade; (1:50 R37110 Invitrogen, Carlsbad, CA) reagent staining was included. Cells were stained with DAPI (1:5000, D3571 Invitrogen, Canada) for 10 min and mounted on glass slides with Mowiol 4-88 (81381-50G Sigma Aldrich, Germany). The embedding was done in a FDM 3D printed chamber, slightly higher than the reinforced scaffold, and glued to a glass slide using nail lacquer, whilst the top was also sealed with glass and lacquer. This was done to easily embed the whole scaffolds and remove all bubbles.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eLive Dead staining of U87 cells and primary astrocytes:\u003c/em\u003e The staining was performed at day 1 and day 7 post-seeding at 21 \u0026deg;C for 20 min with Calcein-AM (2\u0026times;10\u003csup\u003e\u0026minus;6\u003c/sup\u003e M, green/living cells; Thermo Fisher Scientific, Waltham, MA) and Ethidium Homodimer (2\u0026times;10\u003csup\u003e\u0026minus;6\u003c/sup\u003e M, red/dead cells; Sigma-Aldrich, St. Louis, MO) diluted in PBS and incubated for 20 min.\u003c/p\u003e\n\u003cp\u003e\u003cem\u003eConfocal Microscopy and Image Acquisition:\u003c/em\u003e Samples were imaged using an inverted Olympus IX81 microscope equipped with an Olympus FV1000 confocal laser scanning system, a FVD10 SPD spectral detector, and diode lasers of 405 nm (DAPI), 473 nm (Alexa488) and 559 nm (Cy3) (Olympus, Tokyo, Japan). All images shown were acquired using an Olympus UPLSAPO 10\u0026times; (air, numerical aperture 0.4) or Olympus UPLFLN 40x (oil, numerical aperture: 1.3) and were processed using ImageJ/Fiji 1 and Imaris 7.7.2 (Oxford Instrumentals, Abingdon, UK). For cell viability z-stacks of about 2\u0026ndash;3.52 \u0026micro;m step size throughout each sample were acquired. Imaris was used for 3D reconstruction, video generation and reconstruction of the z-stack images to quantitatively analyze live and dead cell numbers The Spots function was used to determine the live/dead ratio. 5 image stacks per experimental condition were analyzed (N = 3). Dynamic range adjustments and projections were done with ImageJ/Fiji Software.\u003c/p\u003e\n\u003cp\u003e\u003cstrong\u003e2.4. Statistical analysis\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eGraphPad Prism 8.3.0 (Graphpad Software, San Diego, CA, USA) was used to calculate mean values, standard deviation (SD), standard error of the mean (SEM), and values for statistical significance. Statistical significance was estimated *p \u0026lt; 0.05 using two-way ANOVA.\u003c/p\u003e"},{"header":"Declarations","content":"\u003cp\u003e\u003cstrong\u003eAcknowledgements\u003c/strong\u003e\u003c/p\u003e\n\u003cp\u003eThis work was supported by the German Research Foundation (DFG, Deutsche Forschungsgemeinschaft) under project number 326998133, within the framework of the Collaborative Research Center/Transregio 225 (SFB/TRR 225) \u0026quot;Biofabrication.\u0026quot; The involved subprojects are A07 (PIs: Gregor Lang, Natascha Sch\u0026auml;fer, and Dirk Schubert), C06 (PI: Taufiq Ahmad), and C05 (PI: Carmen Villmann). Additionally, we acknowledge funding from the DFG Priority Programme SPP 2416, CodeChi, under project number 525934737 (PIs: Sarah Zwingelberg and Gregor Lang). We extend our gratitude to the Graduate School of Life Sciences (GSLS) at the University of W\u0026uuml;rzburg for their support of our Ph.D. students. Additionally, the authors express their thanks to Dr. Thorsten Keller for providing the electrospun PCL/gelatin membrane and to Judith Friedlein for conducting SEM imaging.\u003c/p\u003e\n\u003cp\u003eReceived: ((will be filled in by the editorial staff))\u003c/p\u003e\n\u003cp\u003eRevised: ((will be filled in by the editorial staff))\u003c/p\u003e\n\u003cp\u003ePublished online: ((will be filled in by the editorial staff))\u003c/p\u003e"},{"header":"References","content":"\u003col\u003e\n\u003cli\u003eGill, A. S., Sood, M., Deol, P. K. \u0026amp; Kaur, I. P. 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They have, however, so far been restricted to small scale production in research labs with high batch-to-batch variation. They are commonly produced via electrospinning or melt electro-writing and their delicate nature poses obstacles in detachment, storage, and transportation. This study focuses on overcoming challenges in the high throughput production and practical handling, introducing new methods to reproducibly prepare such scaffolds suitable for quantitative cell culture applications. Attention is given to the seamless handling and transfer of samples without compromising structural integrity. Challenges in detaching fibers without damage as well as storage, and transport are addressed. Cell culture studies demonstrate the methodological advantages, emphasizing the potential for standardized testing and biological readouts of these fiber materials. The developed methods are applicable across various electrospinning and melt electro-writing approaches and can essentially contribute to their utilization in laboratory research and commercial applications.\u003c/p\u003e","manuscriptTitle":"Streamlining the highly reproducible fabrication of fibrous biomedical specimens towards standardization and high throughput","msid":"","msnumber":"","nonDraftVersions":[{"code":1,"date":"2024-03-22 11:14:35","doi":"10.21203/rs.3.rs-4101827/v1","editorialEvents":[{"type":"communityComments","content":0}],"status":"published","journal":{"display":true,"email":"[email protected]","identity":"researchsquare","isNatureJournal":false,"hasQc":true,"allowDirectSubmit":true,"externalIdentity":"","sideBox":"","snPcode":"","submissionUrl":"/submission","title":"Research Square","twitterHandle":"researchsquare","acdcEnabled":true,"dfaEnabled":false,"editorialSystem":"","reportingPortfolio":"","inReviewEnabled":false,"inReviewRevisionsEnabled":true}}],"origin":"","ownerIdentity":"90166d71-85e7-49d6-a653-6e956a9cbc40","owner":[],"postedDate":"March 22nd, 2024","published":true,"recentEditorialEvents":[],"rejectedJournal":[],"revision":"","amendment":"","status":"published-in-journal","subjectAreas":[{"id":29457437,"name":"Health sciences/Medical research/Translational research"},{"id":29457438,"name":"Health sciences/Medical research/Preclinical research"},{"id":29457439,"name":"Biological sciences/Biological techniques/High-throughput screening"},{"id":29457440,"name":"Biological sciences/Biotechnology/Nanobiotechnology/Nanofabrication and nanopatterning"},{"id":29457441,"name":"Biological sciences/Biotechnology/Biomaterials"}],"tags":[],"updatedAt":"2024-12-18T19:24:34+00:00","versionOfRecord":{"articleIdentity":"rs-4101827","link":"https://doi.org/10.1002/adhm.202402527","journal":{"identity":"advanced-healthcare-materials","isVorOnly":true,"title":"Advanced Healthcare Materials"},"publishedOn":"2024-12-15 00:00:00","publishedOnDateReadable":"December 15th, 2024"},"versionCreatedAt":"2024-03-22 11:14:35","video":"","vorDoi":"10.1002/adhm.202402527","vorDoiUrl":"https://doi.org/10.1002/adhm.202402527","workflowStages":[]},"version":"v1","identity":"rs-4101827","journalConfig":"researchsquare"},"__N_SSP":true},"page":"/article/[identity]/[[...version]]","query":{"redirect":"/article/rs-4101827","identity":"rs-4101827","version":["v1"]},"buildId":"8U1c8b4HqxoKbykW_rLl7","isFallback":false,"isExperimentalCompile":false,"dynamicIds":[84888],"gssp":true,"scriptLoader":[]}