Biofabrication

Several recent advances in microphysiological systems (MPSs) or organ-on-chip technology have demonstrated its potential for replacing traditional in vitro and animal models in the coming years. Despite the physiological relevance and cost-effectiveness of organ chips, there are several hurdles that must be overcome for widespread adoption for biological studies. Many shortcomings of manufacturing and scalability have been overcome by a transition from PDMS to thermoplastics. However, challenges have arisen in these sealed, brittle systems related to end-point tissue analyses, harvest, and high-resolution imaging, which is particularly difficult for multi-layer organ chips. Here, we present low-cost organ chips that are fluidically sealed but demountable, fabricated using a cut-and-assemble method without the need for cleanroom technologies. We have validated the capabilities of this method by demonstrating the culture of human aortic smooth muscle cells and induced pluripotent stem cell-derived neural cells, encapsulated in gelatin methacryloyl (GelMA) hydrogel on chip, for up to 27 days. The 3D culture layer of the organ chip was removed, and high-resolution images were obtained via immunostaining. Furthermore, these organ chips facilitate rapid redesign and manufacture for alternative tissue and/or interface systems. To our knowledge, this is the first innervated organ chip with multiple removable cell culture layers, as well as the first humanized nerve-artery model that includes a three-dimensional hydrogel culture. In future work, these unique features of our platform can be utilized for investigating the crosstalk mechanisms between different cell types in co-culture. Impact StatementWe present here a new method for fabricating low-cost demountable organ-on-a-chip platforms. This method leverages our recent cut & assemble method for layered 3D organ chips comprised of gas impermeable thermoplastics.

Organ-on-chip (OOC) technologies, also called microphysi-ological systems (MPS), offer dynamic microenvironments that improve upon static culture systems, yet widespread adoption has been hindered by fabrication complexity, reliance on poly-dimethylsiloxane (PDMS), and limited modularity. Here, we present a modular MPS platform designed for ease of use, re-producibility, and broad applicability. The system comprises layered elastomeric inserts for dual monolayer cell culture, which is clamped within a reusable acrylic cassette for perfusion studies. This enables researchers to decouple model establishment from flow experiments and streamline their work-flows. We validated the system using dual epithelial and en-dothelial cell co-culture under static and perfused conditions, including shear-induced alignment of HUVECs. Material testing confirmed biocompatibility, while vinyl cutting reproducibility demonstrated high manufacturing fidelity. The platform reliably supported long-term culture (up to 14 days), and the open insert format facilitated uniform seeding and imaging access. This approach enables parallelized experimentation, minimizes pump usage, and is well-suited for labs without microfabrication infrastructure. By combining fabrication flexibility with biological robustness, this work establishes a generalizable platform for modular tissue-chip development adapted to diverse organ systems and serves as a foundational framework for democratizing advanced in vitro model systems. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=159 SRC="FIGDIR/small/651503v1_ufig1.gif" ALT="Figure 1"> View larger version (35K): org.highwire.dtl.DTLVardef@1e5fed3org.highwire.dtl.DTLVardef@bcf27eorg.highwire.dtl.DTLVardef@d46f33org.highwire.dtl.DTLVardef@d0912f_HPS_FORMAT_FIGEXP M_FIG C_FIG

Neural organoids (NOs), also known as brain organoids, are derived from human-induced pluripotent stem cells and are Microphysiological Systems (MPS) of the brain that can recapitulate key aspects of neurodevelopment. They enable in vitro studies of brain development and disease mechanisms, providing disease models for various neurodegenerative or neurodevelopmental/degenerative disorders like Alzheimers disease, microcephaly, and autism. There are many protocols to generate NOs with different complexities and sizes, varying from 400 m to several mm in diameter, with a starvation-induced necrotic core eventually forming depending on the diameter and culture conditions. Thus, they can benefit from vascularization and more optimal culture conditions. There have been several attempts to decrease necrosis while growing larger NOs, such as by using orbital shaking or 2D/3D microfluidic chips, but only with limited success. In this study, we describe a 3D finite element model to simulate O2 starvation-induced necrosis in NOs using the Damkohler Number (Da) and the Michaelis-Menten kinetics. We measured the necrotic areas in NOs using fluorescent imaging and used them to calibrate the model with a specific Da. Using these calibrated values, we systematically compared simulations of different NO culture methods--static, orbital shaking, and microfluidic flow around organoids--highlighting their relative impacts on nutrient diffusion and necrosis. We observed that these culture strategies cannot prevent necrosis beyond a diameter of [~]800 m. Based on these findings, we propose that 3D spatial perfusion, achieved through uniformly distributed fluidic capillaries within the NO, could significantly reduce necrosis. We conducted parametric studies on capillary spacing, density, and layout. Our calibrated model offers insights for designing next-generation microfabricated bioreactors and culture devices, not just for NOs but also for all 3D tissue engineering and organoid research.

Three-dimensional (3D) cell culture systems rely on the manipulation of a biologically derived matrix, typically soluble Basement Membrane Extract (sBME), in which cells or cellular aggregates, such as organoids, are suspended. This matrix provides mechanobiological support, promoting cellular processes. However, the handling of sBME-based matrices containing cellular constructs poses significant challenges due to their rheological properties. We developed an integrated bioprinting system to surpass the conventional pipetting, seeding and culture in multiwell plates. The system combines a fluidic cartridge with innovative 3D-printed biocompatible culture tools designed to host and preserve high-throughput microcultures of Patient-Derived Organoids (PDOs) in sBME. The miniaturized hanging-drop configuration enables extended culture periods and high-throughput imaging screenings. This comprehensive approach overcomes common issues associated with sBME, including sedimentation of cellular aggregates, premature gelation, and structural collapse, which negatively impact culture quality and reproducibility throughout the entire 3D culture workflow, from seeding to culture maintenance, and post-culture analyses. O_FIG O_LINKSMALLFIG WIDTH=200 HEIGHT=133 SRC="FIGDIR/small/678315v1_ufig1.gif" ALT="Figure 1"> View larger version (24K): org.highwire.dtl.DTLVardef@19f976eorg.highwire.dtl.DTLVardef@8eeefcorg.highwire.dtl.DTLVardef@1ebe6c7org.highwire.dtl.DTLVardef@7c4403_HPS_FORMAT_FIGEXP M_FIG C_FIG Highlights- Miniaturized 3D hanging-drop matrix-embedded organoid culture in a 384-well plate - Custom cartridge enables homogeneous bioprinting of organoids in sBME-based matrix - 3D-printed tools support compact, scalable multiwell culture systems - System suited for miniaturized culture organoids for high-throughput drug screening - Scalable miniaturized culture system for extended periods of time

Automated technologies are attractive for enhancing a robust manufacturing of tissue engineered products for clinical translation. In this work, we present an automation strategy using a robotics platform for media changes of cartilaginous microtissues cultured in static microwell platforms. We use an automated image analysis pipeline to extract microtissue displacements and morphological features, which serve as input for statistical factor analysis. To minimize microtissue displacement and suspension leading to uncontrolled fusion, we performed a mixed factorial DoE on liquid handling parameters for large and small microwell platforms. As a result, 144 images, with 51 471 spheroids could be processed automatically. The automated imaging workflow takes 2 minutes per image, and it can be implemented for on-line monitoring of microtissues, thus allowing informed decision making during manufacturing. We found that time in culture is the main factor for microtissue displacements, explaining 10 % of the displacements. Aspiration and dispension speed were not significant at manual speeds or beyond, with an effect size of 1 %. We defined optimal needle placement and depth for automated media changes and we suggest that robotic plate handling could improve the yield and homogeneity in size of microtissue cultures. After three weeks culture, increased expression of COL2A1 confirmed chondrogenic differentiation and RUNX2 shows no osteogenic specification. Histological analysis showed the secretion of cartilaginous extracellular matrix. Furthermore, microtissue-based implants were capable of forming mineralized tissues and bone after four weeks of ectopic implantation in nude mice. We demonstrate the development of an integrated bioprocess for culturing and manipulation of cartilaginous microtissues. We anticipate the progressive substitution of manual operations with automated solutions for manufacturing of microtissue-based living implants.

Cell culture devices, such as microwells and microfluidic chips, are designed to increase the complexity of cell-based models whilst retaining control over culture conditions and have become indispensable platforms for biological systems modelling. From microtopography, microwells, plating devices and microfluidic systems to larger constructs for specific applications such as live imaging chamber slides, a wide variety of culture devices with different geometries have become indispensable in biology laboratories. However, while their application in biological projects is increasing exponentially, due to a combination of the techniques and tools required for their manufacture, and the physical science background sometimes needed, the design and fabrication of such devices directly by biological labs remains a relatively high investment in terms of costs, use of facilities, needed collaborations and time. Whilst commercially available systems are available, these are also often costly, and importantly lack the potential for customisation by each single lab. This combination of factors still limits widespread application of microfabricated custom devices in most biological wet labs. Capitalising on recent important advancements in the fields of bioengineering and microfabrication, and taking advantage of low-cost, high-resolution desktop resin 3D printers combined with PDMS soft lithography, we have developed an optimised low-cost and highly reproducible microfabrication pipeline, capable of generating a wide variety of customisable devices for cell culture and tissue engineering in an easy, fast reproducible way for a fraction of the cost of conventional microfabrication or commercial alternatives. This protocol is designed specifically to be a resource for biological labs with little to none prior exposure to these fields technique and enables the manufacture of complex devices across the {micro}m to cm scale. We provide a ready-to-go pipeline for the efficient treatment of resin-based 3D printed constructs for PDMS curing, using a combination of curing steps, washes and surface treatments. Together with the extensive characterisation of the fabrication pipeline, we show the utilization of this system to a variety of applications and use cases relevant to biological experiments, ranging from micro topographies for cell alignments to complex multi-part hydrogel culturing systems. This methodology can be easily adopted by any wet lab, irrespective of prior expertise or resource availability and will enable the wide adoption of tailored microfabricated devices across many fields of biology.

Melt electrowriting (MEW) is an additive manufacturing technique characterized by its ability to fabricate micronscale fibers from molten polymers into highly controlled 3D microfiber scaffolds. This emerging technique is gaining traction in tissue engineering and biofabrication research, however limitations in the ability to develop advanced coding to program MEW printers to fabricate scaffolds with complex fiber architectures has inhibited the development of structures with tunable and biomimetic mechanical properties. This study reports a series of non-straight scaffold architectures with combinations of independently controlled X & Y fiber spacing, corrections for MEW jet lag, and characterizations of their influences on scaffold mechanics. Polycaprolactone scaffolds with an elastic modulus ranging from 0.3 to 7.3 MPa were fabricated utilizing scaffolds manufactured from 5 layers of 55 m fibers. The inclusion of scaffold design corrections in the gcode to compensate for decreasing deposition accuracy with increasing layer height enabled us to correct for discontinuous stress-strain mechanics and improved scaffold fabrication reproducibility. This study provides a comparison between a series of highly reproducible MEW scaffold architectures with non-straight fibers compared to the common crosshatch design to inform the development of more biomimetic scaffolds applicable to a variety of clinical applications. It further illustrates the significant effect toolpath correction has on reducing poor stress-strain mechanics, therefore improving the control, reproducibility, and biomimetic capacity of the MEW technique.

Recent technological advances in the field of Additive Manufacturing (AM) and the increasing need in Regenerative Medicine (RM) for devices that better and better mimic native tissues architecture are showing limitations in the current scaffolds fabrication techniques. A switch from the typical layer-by-layer approach is needed to achieve precise control on fibers orientation and pores dimension and morphology. In this work a new AM apparatus, the RAVEN (Robot-Assisted Volumetric ExtrusioN) system, is presented. RAVEN is based on a 7-DOF robotic arm and an FDM extruder and allows for volumetric extrusion of polymeric filaments. The development process, namely the robotic motion optimization, the optimization towards small-scale trajectories, the custom-made hardware/software interfaces, and the different printing capabilities are hereby presented. The successful results are promising towards future advanced applications such as in vivo bioprinting, in which the ability of the robot to change its configuration while printing will be crucial.

Controlled volume microliter cell-laden droplet bioprinting is important for precise biologics deposition, reliably replicating 3D microtissue environments for building cell aggregates or organoids. To achieve this, we propose an innovative machine-learning approach to predict cell-laden droplet volumes according to input parameters. We developed a novel bioprinting platform capable of collecting high-throughput droplet images and generating an extensive dataset for training machine learning and deep learning algorithms. Our research compared the performance of three machine learning and two deep learning algorithms that predict droplet volume based on numerous bioprinting parameters. By adjusting bioink viscosity, nozzle size, printing time, printing pressure, and cell concentration as input parameters, we precisely could control droplet sizes, ranging from 0.1 {micro}L to 50 {micro}L in volume. We utilized a hydrogel precursor composed of 5% gelatin methacrylate and a mixture of 0.5% and 1% alginate, respectively. Additionally, we optimized the cell bioprinting process using green fluorescent protein-tagged 3T3 fibroblast cells. These models demonstrated superior predictive accuracy and revealed the interrelationships among parameters while taking minimal time for training and testing. This method promises to advance the mass production of organoids and microtissues with precise volume control for various biomedical applications.

Inexpensive stereolithography (SLA) 3D printing enables rapid prototyping of resin molds for polydimethylsiloxane (PDMS) soft lithography and organ chip fabrication, but geometric distortion and surface roughness of SLA resins can impede the development of adaptable manufacturing workflows. This study reports post-processing procedures for manufacturing SLA-printed molds built with a Formlabs F3 printer that produce fully cured, flat, patently bonded, and optically clear PDMS organ chips. User injection loading tests with iterated guide structure designs were conducted to achieve engineering reduction to practice of milliscale membrane-free organ chips (MFOC), defined as reproducible loading of aqueous solutions without failure of surface tension-based liquid patterning. The optimized manufacturing workflow was applied to further engineer milliscale MFOC for specific applications in modeling vascular physiology and pathobiology. The open lateral interfaces of bulk tissues seeded in MFOC facilitate the formation of anastomoses with internal vasculature to create milliscale perfusable vascular beds. After optimizing bulk tissue vasculogenesis in MFOC, we developed a method for seeding the bulk tissue interfaces with a confluent endothelium to stimulate self-assembly of perfusable anastomoses with the internal vasculature. Rocker- and pump-based flow-conditioning protocols were tested to engineer enhanced barrier function of the perfusable internal vasculature. Modularity of the MFOC design enabled creation of a multi-organ device that was used to model decaying gradients of cancer-associated vascular inflammation in organ compartments positioned at increasing distances from a tumor compartment. These easily adaptable methods for designing and fabricating vascularized microphysiological systems can accelerate their adoption in a diverse range of preclinical laboratory settings.

Biological research groups may face a high barrier to entry when constructing custom 3D cell culture devices to investigate multi-tissue interactions in vitro. Standard fabrication methods such as lithography, etching, or molding are expensive and require specialized equipment and expertise. To address this, we developed an accessible approach for producing polyethylene glycol (PEG)-based cell culture devices using stereolithography (SLA) 3D printing with a polydimethylsiloxane (PDMS) intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, the final device swells to predictable dimensions and retains its shape for at least 10 days. We used this approach to develop a human pluripotent stem cell (hPSC)-derived neural spheroid outgrowth model that supports directed neurite extension over 14 days. Together, this method provides a highly customizable, affordable platform for rapid fabrication of PEG-based microphysiological systems (MPS) for diverse tissue engineering applications. ImpactAs biomedical labs work to complement animal models with tissue-engineered MPSs, there is a growing need for low-cost, rapid, and iterative fabrication workflows. We developed a pipeline combining 3D printing, a PDMS intermediate mold, and PEG casting, avoiding the need for specialized photolithography. The resulting devices support stable, nutrient-permissive cell culture while allowing control over device dimensions and customizable channel or compartment configurations. We demonstrate its utility with reprogrammed hPSC-derived neurons, which remain challenging to support sustained neurite outgrowth in engineered models. This workflow expands access to cell culture device fabrication for MPSs across a broader range of biological laboratories.

Vascular smooth muscle cells (vSMCs) are one of the essential cell types in blood vessel walls. A significant vSMC phenotype characteristic is that they collectively wrap around the outer layer of the healthy blood vessels with spindle-like morphology and help maintain the vascular tones and regulate the blood flow. Both physiological and biomedical research are impeded by the standard 2D cell culture approaches which do not create in vivo like microenvironment. Here, we systematically investigated the vSMCs culturing within 3D printed geometrical constraints and on printed microfilaments. Based on these models, we demonstrate a simple bioprinting approach for fast manufacturing vessel architectures with micro-grooved surfaces for vSMCs alignment. We validated that the vSMCs cultured on the printed vessel with microfilaments (VWMF) present a more physiologically relevant morphological phenotype and gene expression profile, and they are considerably more active in wound healing and ischemia than conventional planarly cultured vSMCs.

Bioreactors for reseeding of decellularized lung scaffolds have evolved with a wide variety of advancements. These include biomimetic mechanical stimulation, constant nutrient flow, multi-output monitoring, and large mammal scaling. Although dynamic bioreactors are not new to the field of bioengineered lungs, ideal conditions during cell seeding have not been extensively studied or controlled. To address the lack of cell dispersal in traditional seeding methods, we have designed a two-step bioreactor. The first step rotates a seeded lung every 20 minutes at different angles to ensure 20 percent of cells are anchored to a particular location based on the known rate of attachment. The second step involves perfusion culture to ensure nutrient dispersion and cellular growth. Compared to statically seeded lungs followed by conventional perfusion, rotationally seeded lungs followed by perfusion had significantly increased dsDNA content and more uniform cellular distribution. This new bioreactor system will aid in recellularizing the lung and other geometrically complex organs for tissue engineering.

3D cell culture models have gained popularity in recent years as an alternative to animal and 2D cell culture models for pharmaceutical testing and disease modeling. Polydimethylsiloxane (PDMS) is a cost-effective and accessible molding material for 3D cultures; however, routine PDMS molding may not be appropriate for extended culture of contractile and metabolically active tissues. Failures can include loss of culture adhesion to the PDMS mold and limited culture surfaces for nutrient and waste diffusion. In this study, we evaluated PDMS molding materials and surface treatments for highly contractile and metabolically active 3D cell cultures. PDMS functionalized with polydopamine allowed for extended culture duration (14.8 {+/-} 3.97 days) when compared to polyethylamine/glutaraldehyde functionalization (6.94 {+/-} 2.74 days); Additionally, porous PDMS extended culture duration (16.7 {+/-} 3.51 days) compared to smooth PDMS (6.33 {+/-} 2.05 days) after treatment with TGF-{beta}2 to increase culture contraction. Porous PDMS additionally allowed for large (13 mm tall x 8 mm diameter) constructs to be fed by diffusion through the mold, resulting in increased cell density (0.0210 {+/-} 0.0049 mean nuclear fraction) compared to controls (0.0045 {+/-} 0.0016 mean nuclear fraction). As a practical demonstration of the flexibility of porous PDMS, we engineered a vascular bioartificial muscle model (VBAM) and demonstrated extended culture of VBAMs anchored with porous PDMS posts. Using this model, we assessed the effect of feeding frequency on VBAM cellularity. Feeding 3x/week significantly increased nuclear fraction at multiple tissue depths relative to 2x/day. VBAM maturation was similarly improved in 3x/week feeding as measured by nuclear alignment (23.49{degrees} {+/-} 3.644) and nuclear aspect ratio (2.274 {+/-} 0.0643) relative to 2x/day (35.93{degrees} {+/-} 2.942) and (1.371 {+/-} 0.1127), respectively. The described techniques are designed to be simple and easy to implement with minimal training or expense, improving access to dense and/or metabolically active 3D cell culture models.

Bespoke cell culture devices are essential for tissue engineering applications. Traditional manufacturing methods for cell culture accessories involve injection molding and machining, which are too costly and time-consuming to implement for producing custom designs in small batches, and/or while testing the usefulness of a new design before mass producing it. Materials typically used for rapid design iteration, like poly(dimethylsiloxane) (PDMS) elastomers, surmount this issue but present new challenges of affinity for hydrophobic small molecules and sub-optimal interactions with sensitive cell types. Here, we propose polycarbonate (PC) thermoforming as a solution for creating customized transparent and autoclavable accessories. We demonstrate that optimized preheating of PC overcomes issues with bubbling during thermoforming. The use of high heat deflection temperature (HDT) resins allows these PC devices to be thermoformed off molds created by Digital Light Processing (DLP) 3D prints, enabling rapid prototyping of the PC. Using this approach, we fabricated custom PC well plate inserts. These inserts combine many advantages of tissue culture polystyrene (negligible absorption of hydrophobic molecules, transparency, rigidity) and elastomers (ease of creating bespoke devices, ability to be sterilized by autoclaving) and are compatible with a variety of cell biology applications, including human induced pluripotent stem cells (iPSC) culture. PC inserts also supported iPSC differentiation into cardiomyocytes (iPS-CM) and micro-patterning of iPS-CM into cardiospheres. This low cost, customizable approach holds promise for a variety of bioengineering applications.

Pluripotent stem cells represent a potentially unlimited cell source for the fabrication of human bioartificial tissues to study and treat degenerative conditions such as type 1 diabetes. Alginate is widely used for mammalian cell immobilization and the primary hydrogel studied for pancreatic islet encapsulation. Rheological properties of alginate solutions or fully gelled forms are unsuitable as support matrix for embedded 3D printing. We describe partially gelled self-healing alginate formulations tuned for embedded 3D printing. Perfusable multi-plane hierarchical networks branching into 10 parallel channels, obtained by 3D printing of Pluronic F127 into the alginate support, show high fidelity to computer-assisted models. Therapeutic {beta}-cell doses (40x106 cells/mL) within centimeter-thick perfusable constructs remained viable for at least 1 week of culture under flow, with rapid insulin secretion detected upon glucose challenges. Stem cell-derived islet clusters cultured in 5-channel contructs for 25 days differentiated towards functional insulin-expressing cells. We describe a novel approach to generate cm-scale perfusable endocrine pancreatic constructs using sacrificial embedded 3D printing into alginate. This approach offers an adaptable platform to engineer perfusable cm-scale functional endocrine pancreatic tissues and potentially other vascularized bioartificial tissues.

Human induced pluripotent stem cell-derived cardiomyocytes (hiPSC-CMs) offer numerous advantages as a biological model, yet their inherent immaturity compared to adult cardiomyocytes poses significant limitations. This study addresses hiPSC-CM immaturity by introducing a novel physiologically relevant micropatterned substrate for long-term culture and maturation. A novel microfabrication technique combining laser etching and casting creates a micropatterned polydimethylsiloxane (PDMS) substrate with varying stiffness, from 2 to 50 kPa, mimicking healthy and fibrotic cardiac tissue, respectively. Platinum electrodes integrated into the cell culture chamber enabled pacing of cells at various frequencies. Subsequently, cells were transferred to the incubator for time-course analysis, ensuring contamination-free conditions. Cell contractility, cytosolic Ca2+ transient, sarcomere orientation, distribution, and nucleus aspect ratio are analyzed in a 2D hiPSC-CM monolayer up to 90 days post-replating in relation to substrate micropattern dimensions. Culturing hiPSC-CMs for three weeks on a micropatterned PDMS substrate (2.5-5 {micro}m deep, 20 {micro}m center-to-center spacing of grooves, 2-5 kPa stiffness) emerges as optimal for cardiomyocyte alignment, nucleus aspect ratio, contractility, and cytosolic Ca2+ transient. The study provides significant insights into substrate stiffness effects on hiPSC-CM contractility and Ca2+ transient at immature and mature states. Maximum contractility and fastest Ca2+ transient kinetics occur in mature hiPSC-CMs cultured for two to four weeks, with the optimum at three weeks, on a soft micropatterned PDMS substrate. This new substrate offers a promising platform for disease modeling and therapeutic interventions.

A major limitation of islet transplantation as a therapy for treating Type 1 Diabetes is eventual graft failure, which can be partially attributed to islet cell death. When cultured in vitro, cells in the center of large islets show increased necrosis and exhibit decreased viability and insulin secretion compared to smaller islets. Given the necessity of {beta}-cell-to-{beta}-cell coupling for the physiological response to glucose, a technique to re-aggregate primary islet cells or cells derived from progenitor cells into small clusters of defined sizes may prove advantageous for promoting function upon transplantation. Here, hydrogel microwell arrays were utilized to generate 3-dimensional pseudo-islets from primary murine islets. Pseudo-islets ranged from 50 to 100 m in diameter as controlled through the microwell dimensions, and contained {beta}-, -, and {delta}-cells with ratios similar to those in whole murine islets. Over two weeks in culture, pseudo-islets remained highly viable and responsive to glucose. Intracellular calcium flux showed more robust and coordinated dynamics at high glucose and decreased activity at low glucose compared to age-matched wild-type islets. Therefore, microwell devices can control the aggregation of cells isolated from primary islets to produce islet-like clusters that are functionally similar to freshly isolated islets, and may provide a technique to create improved cellular therapies for Type 1 Diabetes.

This paper introduces the concept of continuous chaotic printing, i.e., the use of chaotic flows for deterministic and continuous fabrication of fibers with internal multilayered micro-or nanostructures. Two free-flowing materials are coextruded through a printhead containing a miniaturized Kenics static mixer (KSM) composed of multiple helicoidal elements. This produces a fiber with a well-defined internal multilayer microarchitecture at high speeds (>1.0 m min-1). The number of mixing elements and the printhead diameter determine the number and thickness of the internal lamellae, which are generated according to successive bifurcations that yield a vast amount of inter-material surface area (~102 cm2 cm3) and high resolution features (~10 m). In an exciting further development, we demonstrate a scale-down of the microstructure by 3 orders of magnitude, to the nanoscale level (~10 nm), by feeding the output of a continuous chaotic 3D printhead into an electrospinner. Comparison of experimental and computational results demonstrates the robust and predictable output and performance of continuous chaotic 3D printing. The simplicity and high resolution of continuous chaotic printing strongly supports its potential use in novel applications, including--but not limited to--bioprinting of multi-scale tissue-like structures, modeling of bacterial communities, and fabrication of smart multi-material and multilayered constructs.

Advances in cancer treatments have greatly improved pediatric cancer survival rates, leading to quality of life considerations and in particular fertility restoration. Accordingly, pre-pubertal patients have the option to cryopreserve testicular tissue for experimental restorative therapies, including in vitro spermatogenesis, wherein testicular tissue is engineered in vitro and spermatozoa are collected for in vitro fertilization (IVF). Current in vitro systems have been unable to reliably support the generation of spermatozoa from human testicular tissues, likely due to the inability for the dissociated testicular cells to recreate the native architecture of testicular tissue found in vivo. Recent advances in 3-D bioprinting can place cells into geometries at fine resolutions comparable to microarchitectures found in native tissues, and therefore hold promise as a tool for the development of a biomimetic in vitro system for human spermatogenesis. This study assessed the utility of bioprinting technology to recreate the precise architecture of testicular tissue and corresponding spermatogenesis for the first time. We printed testicular cell-laden hollow microtubules at similar resolutions to seminiferous tubules, and compared the results to testicular organoids. We show that the human testicular cells retain their viability and functionality post-printing, and illustrate an intrinsic ability to reorganize into their native cytoarchitecture. This study provides a proof of concept for the use of 3-D bioprinting technology as a tool to create biomimetic human testicular tissues.