Biofabrication最新文献

Techniques for fabricating cell sheets with two-dimensional (2D) patterns have advanced significantly over the years. However, creating cell sheets featuring three-dimensional (3D) raised textures that mimic the complex architecture of natural tissues continues to present a considerable challenge. This study introduces a versatile methodology for fabricating cell sheets with both 2D patterns and 3D microstructures, utilizing picosecond laser-induced microgroove-patterned polystyrene (PS) culture dishes. This technology leverages the sequential interaction of the laser with the photosensitive coating and the PS substrate to create microgrooves with exceptional precision in both width and depth. Both direct ablation and coating-assisted ablation result in patterned culture dishes that demonstrate excellent cell compatibility, an absence of cytotoxicity, and the ability to regulate cell proliferation. The patterned PS dishes create distinct 3D microenvironments that guide cell contact and adhesion arrangements, thereby modulating gene expression and protein secretion in normal human dermal fibroblasts. Notably, key proteins such as type I alpha1 collagen (Col-1), type VI alpha1 collagen (Col-6), Elastin, fibronectin, and matrix metalloproteinase 2 (MMP-2) are significantly influenced by the structure of pattern. Furthermore, cell sheets with raised textures (CSRTs) can be detached from the patterned PS culture dishes while preserving their 3D structure for over 72 h, with structural longevity dependent on feature size. To our knowledge, this study represents the first successful fabrication of CSRT using a laser-induced micro-patterning technique. This approach provides foundational insights into the development of biomimetic tissues for regenerative medicine and advancedin vitromodels, offering a promising platform for future applications in tissue engineering and biomedical research.

Microelectrode arrays (MEAs) can be used to record extracellular field potentials of cells, enabling investigations on neural or cardiac cellular electrical activity. However, conventionally used 2D cell monolayers cannot recapitulate the 3D microenvironment of in vivo tissue. Therefore, cells are grown in 3D cultures that mimic the architectural and functional aspects of human organs. Microelectrode arrays that support such 3D structures are of increasing importance, but their fabrication often relies on advanced cleanroom techniques. Here, we present a fast and straightforward prototyping technique of a thin-film porous microelectrode array fabricated by conformal coatings and laser ablation. The absence of photolithography processes allows the microelectrode array to be directly fabricated as a 3D structure. This advantage was exploited by manufacturing 3D, well-shaped MEAs to host cortical organoids for extracellular signal recordings. The 3D-printing-based fabrication of the wells enables to tune the size of the MEA according to the size of the organoid. The proposed well-shaped MEAs enable easy handling and secure organoid placement by physically retaining the organoid within the well, ensuring direct alignment with underlying electrodes avoiding the detachment issues typically encountered on 2D MEA designs. We present extracellular field potential recordings from both cardiac cells and cortical organoids.

The emergence of SARS-CoV-2 led to a global pandemic with severe respiratory symptoms and substantial extrapulmonary manifestations. Increasing evidence suggests significant cardiovascular complications associated with SARS-CoV-2 infection, which are critical factors in morbidity and mortality. In this study, we assessed the viral infectivity and viral niche of SARS-CoV-2 using our clinically-amenablein vitrocardiac spheroids (CSs), which have previously been demonstrated to be an optimal tool to recapitulate the complex cardiac pathophysiology. We examined the expression profiles of cardiovascular-related disease genes and pathways involved in inflammation, interferon responses, and antiviral defence following infection. Genes associated with apoptosis, chemotaxis, fibrosis, and contractile function exhibited substantial increases, implicating these pathways in the cardiac response to SARS-CoV-2. Furthermore, our 3D rendering analyses using confocal imaging revealed cell-specific effects mediated by the virus by colocalising SARS-CoV-2 nucleocapsid protein with each cell type, supporting the ability of CSs to facilitate viral replication and contributing to the observed phenotypes. Additionally, SARS-CoV-2 could only infect intact CSs, whereas it did not infect individual cell types cultured individually. The unique ability of CSs to model SARS-CoV-2 in the heart may potentially mirror the pathophysiological changes observed in COVID-19-induced cardiac complications. Altogether, our results suggest that CSs offer a valuable tool for dissecting direct host-viral interactions and advancing our understanding of SARS-CoV-2-related cardiac injury. Our findings underscore the utility of CSs in revealing the mechanisms of SARS-CoV-2-induced cardiac damage and provide a basis for further studies into the long-term cardiovascular consequences of SARS-CoV-2.

In this work, we developed a collagenase-responsive hydrogel system to covalently load cancer immunotherapy candidate cowpea mosaic virus (CPMV) using 3D digital light processing bioprinting technology. CPMV was functionalized with norbornene groups (CPMV-NB), which was then bioprinted into hydrogels with 8-arm polyethylene glycol norbornene and a collagenase-cleavable peptide via photoinduced thiol-ene click chemistry. This strategy enabled stable retention of CPMV-NB within the hydrogels and achieved controlled release of CPMV-NB triggered by collagenase. Furthermore, released CPMV-NB retained its immunogenicity to stimulate immune cells.

The severe shortage of donor organs and limitations of current disease models highlight the urgent need for transformative strategies in tissue engineering (TE) and regenerative medicine (RM). Bioprinting has emerged as a powerful approach for creating functional tissues and organs, yet current workflows remain labor-intensive, variable, and challenging to scale. The convergence of artificial intelligence (AI), advanced bioprinting technologies, robotics, biosensing, and cutting-edge biological methods is catalyzing the development of self-driving bioprinting laboratories-a fully integrated, autonomous, closed-loop system capable of designing, fabricating, maturing, and assessing living tissue constructs, as well as supporting seamless transplantation, with minimal human intervention. By integrating autonomous cellular farming, on-demand bioink formulation, intelligent optical and digital reconstruction platforms, AI-driven bioprinting, intelligent bioreactors, and robotic transplantation within a sterile, interconnected ecosystem, such platforms can continuously learn, adapt, and optimize workflows, enabling standardized, scalable tissue manufacturing and facilitating a seamless transition from bench to bedside. This perspective outlines the foundational technologies, opportunities, and challenges for realizing self-driving bioprinting, envisioning a future where intelligent, automated platforms transform TE and RM into a scalable, predictive, and clinically integrated discipline at the forefront of precision medicine.

We present a precision-engineered lung-on-chip platform that replicates the biomechanical and structural features of the human alveolar microenvironment for respiratory disease modeling and therapeutic evaluation. At the core of the device is a thin, suspended hydrogel membrane composed of biologically relevant collagen and elastin, engineered to mimic the dimensions and mechanical fragility of the native alveolar basement membrane. This membrane supports a geometrically defined array of alveolar units, each capable of undergoing finely controlled, physiologically relevant deflections under cyclic mechanical actuation-emulating the subtle deformations that occur during human breathing. To address the challenges posed by the membrane's mechanical fragility and the requirement for accurately controlled micron-scale deflections, the platform is fabricated using precision injection molding. This manufacturing strategy ensures structural integrity and reproducibility, creating a rigid support structure around the suspended hydrogel membrane. The design is integrated into a SBS microwell plate format, facilitating robust fluidic interfacing, consistent cyclic actuation, and medium-throughput operation. Human alveolar epithelial cells and lung fibroblasts are co-cultured on a membrane and subjected to cyclic biomechanical stress that mimics respiratory movements. We demonstrate that cyclic stretching significantly amplifies fibrotic signaling in the presence of transforming growth factor-beta 1 (TGF-β1), evidenced by increased expression of extracellular matrix (ECM) components such as collagen I, collagen III, and fibronectin. Treatment with the anti-fibrotic drug nintedanib reduced expression of ECM proteins and plasminogen activator inhibitor-1 (PAI-1), validating the system's utility for pharmacological testing. This alveolar array-based lung-on-chip system bridges a critical gap between conventionalin vitromodels and the physiological complexity of human lung tissue, offering a robust platform for mechanistic studies and preclinical evaluation in pulmonary fibrosis and related disorders.

The BBB remains a major obstacle to effective treatment of CNS disorders by limiting the entry of most therapeutics into the brain. The hCMEC/D3 is widely used as anin vitromodel to study BBB structure, permeability, and drug transport. In parallel, intranasal administration has gained prominence as a non-invasive route to bypass the BBB and deliver therapeutics directly to the brain via olfactory and trigeminal pathways. This review critically explores how hCMEC/D3 models support the development of intranasal N2B drug delivery strategies. Advances in co-culture systems, 3D constructs, and microfluidic BBB-on-chip platforms have improved the physiological relevance of hCMEC/D3. Integration with nasal epithelial models, including ALI cultures and nasal-on-chip systems, enables simulation of the entire N2B transport route. Emerging delivery systems, including mucoadhesive nanoparticles, ligand-targeted carriers, and prodrugs, are evaluated for their performance in dual-barrierin vitromodels. While progress is evident, challenges remain in translatability and standardisation. Future efforts integrating omics, machine learning, and organ-on-chip technologies will enhance predictive modelling and accelerate CNS drug development.

Craniofacial bone defects, particularly alveolar clefts, pose significant clinical challenges in pediatric patients due to complex anatomy and the limitations of current grafting options. Although autologous bone grafts remain the clinical gold standard, their use is restricted by donor-site morbidity, limited tissue availability, high cost, and risks such as infection, chronic pain, and functional impairment. Decellularized and demineralized bone matrix (DDBM) offers an attractive alternative but lacks controlled drug-release capability and cannot be monitored in real time in patients. To address these limitations, we developed indocyanine green-encapsulated bone-derived nanoparticles (ICG/BPs) from porcine DDBM, combining the intrinsic osteoinductive and osteoconductive properties of DDBM with near-infrared (NIR) imaging functionality. In this study, we fabricated two ICG/BP formulations, crosslinked (X-ICG/BP) and uncrosslinked (UnX-ICG/BP), and compared their in vitro degradation, release profiles, and in vivo performance in a rat model of cavity-type alveolar defects. Crosslinking improved particle stability and prolonged ICG release, and NIR imaging enabled real-time, non-invasive monitoring of particle degradation and retention within the defect. Additionally, both ICG/BP formulations supported bone regeneration, with X-ICG/BPs demonstrating greater regeneration, tissue organization, and vascularization. Overall, these findings highlight the tunability and theranostic potential of ICG/BPs and support their continued development as an image-guided functional biomaterial for craniofacial bone repair. .

Three-dimensional (3D) bioprinting is an emerging strategy for constructing tissues and organsin vitro. Here, we achieved long-term expansion of primary mouse hepatocytes using a defined medium and constructed liver tissue using 3D bioprinting. The 3D-printed liver tissue demonstrated several essential liver functions and was able to prolong the survival of mice with acute liver failure due to extreme hepatectomy afterin vivotransplantation, and the transplanted artificial liver tissue showed distinct functional partitioning. Overall, our results develop a method for long-termin vitroculture of primary hepatocytes and demonstrate the potential of 3D bio-printed liver tissue for clinical translational applications.

Hepatic organoids are potential building blocks for three-dimensional (3D) bioprinting of liver tissue models, as they mimic tissue-level cellular patterning and functions. However, traditional hydrogel-embedded organoid development methods are less dynamic and require harsh retrieval techniques that negatively affect organoid integrity, function, and tissue continuity within bioprinted constructs. In this study, we evaluated the utility of mesenchymal stem cells (MSCs) and a thermoresponsive culture substrate, poly (N-isopropyl acrylamide-co-glycidyl methacrylate) (NGMA), for liver organoid formation and easy retrieval, as well as the potential of such non-invasively harvested organoids for extrusion-based 3D bioprinting. Primary rat liver cells were seeded on MSC-laden NGMA substrate and cocultured for 72 hours under defined conditions. The cells self-organized to form viable and functional liver organoids that detached from the substrate as organoid sheets when exposed to low temperature. Moreover, the organoid sheets and organoid-forming cells were independently encapsulated in methacrylated gelatin bioink to produce liver tissue constructs via extrusion bioprinting. Compared to cell-laden constructs, the organoid-laden construct exhibited higher levels of liver-specific gene expression and tissue functions such as protein synthesis, ammonia detoxification, and drug metabolism. Histological analysis of the organoid-laden construct revealed 'histomimetic' cellular organization and the expression of tissue-specific markers. Overall, the MSC-laden NGMA substrate proved to be an ideal platform for generating intact liver organoids for 3D bioprinting of histomimetic liver tissue models. The methodology established here can be used to develop a reliable liver tissue model for drug testing, disease modeling, and regenerative therapy.