Biofabrication最新文献

Organ-on-a-chip (OoC) systems are microfluidic technologies that replicate human physiology and disease conditionsex vivo, offering a promising alternative to animal models in preclinical drug testing and fundamental biological studies. Traditionally, OoCs systems are fabricated using conventional soft-lithography techniques with polydimethylsiloxane (PDMS) primarily due to its excellent inherent properties, including gas permeability, optical transparency, and biocompatibility. However, PDMS presents several notable shortcomings, most its limited scalability, which have prompted the search for more rapid and scalable fabrication processes. In this study, we present a cost-effective, efficient, and rapid design, development, and prototyping process for a microfluidic tumor-on-a-chip (TOC) platform technology for applications in cancer research and drug screening. Specifically, we present a novel 3D-printed, closed-system TOC device (i.e. Biochip) featuring distinct yet interconnected tumor and stromal regions, separated by an array of trapezoidal microposts, and fabricated with high precision and fidelity. The proposed Biochip was fabricated utilizing vat polymerization with a biocompatible resin and was compared alongside a conventional PDMS-glass (PDMS-G) and PDMS-laminate (PDMS-L) TOCs to evaluate its biological outcomes. The fabricated Biochip supported closed-channel 3D cell culture for testing up to 5 d. Using two triple-negative breast cancer cells, namely SUM-159 and MDA-MB-231, we further assessed and cross compared cellular migration, viability, and morphology across the Biochip, PDMS-G, and PDMS-L platforms. Overall, this work establishes a 3D-printed Biochip as a robust, cost-effective, and time-saving alternative to PDMS-based OoC, and specifically TOC systems.

Stiffening of the extracellular matrix underlying the epithelial cells of the large intestine is associated with aging as well as many diseases. Yet the impact of the stiffened matrix on epithelial physiology remains poorly understood. A 2D and 3D microphysiological model of the large intestine was developed using a collagen scaffold with a physiologic or excessive stiffness (Young's moduli of 2.84 ± 0.85 kPa and 15.9 ± 0.73 kPa) by altering the collagen concentration within the substrate. Diffusion of a 10 and 40 kDa fluorescent dextran was significantly different between the physiologic and stiff scaffold (97.8 vs 79.8 µm2/s [10 kDa] and 68.2 vs 56.8 µm2/s [40 kDa], respectively). When primary human epithelial cells of the large intestine were grown as a 2D monolayer, cultures on the physiologic scaffold grew to a significantly higher density with more proliferative and fewer differentiated cells than cultures on the stiffened scaffold. Three-dimensional crypt arrays were also fabricated with the physiologic and stiff substrates, populated with cells, and a growth factor gradient applied. The cell density, proliferation, and height-to-width ratio was significantly greater for cells on the physiologic scaffold relative to that of cells on the stiffened scaffolds. Placement of a layer of intestinal fibroblasts below the epithelium on the crypt arrays did not mitigate the impact of the stiffened substrate. Bulk-RNA sequencing revealed 378 genes that were significantly upregulated and 385 genes significantly downregulated in the stiffened vs physiologic scaffolds. This work demonstrates that a molded collagen hydrogel can be used to mimic the biophysical characteristics of a stiffened intestinal stroma, recapitulating physiology observed in vivo. This in vitro model of polarized crypts with a tunable underlying substrate will enable an improved understanding of intestinal epithelial cell morphology, stem cell maintenance and lineage allocation within a stiffened environment.

Osteochondral defects are injuries generally affecting to the surface of hyaline cartilage and progressing throughout the tissue until the underlying subchondral bone. The osteochondral unit is a multi-zonal tissue in which cells within each layer have a specific phenotype arising from their differential maturation stages; persistent, proliferative and hypertrophic chondrocytes in the superficial, middle and deep zones of cartilage, respectively, and osteoblast in the subchondral bone. These distinct cells regulate the composition of their microenvironment through sensing the surrounding physicochemical properties, where topography plays a crucial role. Tissue regeneration appears as a great alternative to promote the formation of a durable and functional osteochondral unit, where distinct parameters such as the biomaterial chemistry, mechanical properties or topography can be adjusted to match the native tissue. However, current approaches focus mainly on tuning the first two parameters, omitting the inclusion of topography. Moreover, only few have considered the inclusion of topography on scaffolds and investigated their effect in pre-clinical studies; number that is further reduced when reaching clinical trials. This review summarizes the state of the art in the regeneration of the osteochondral unit through the exploitation of topographical cues, setting into context relevant biological aspects, such as cell adhesion and proliferation, phenotype and deposition of zone-specific extracellular matrix that lead to the formation of a functional tissue.

The large-scale production of mammalian cells, particularly stem cells for clinical applications, remains challenging with existing cell culture technologies such as two-dimensional cell culture flasks or three-dimensional stirred tank bioreactors. Current methods have issues such as excessive cell aggregation and significant shear stress-induced cell death, resulting in low cell yield, unacceptable batch-to-batch variation, high production costs, and difficulties in scaling up. We hypothesize that creating a cell-friendly microenvironment that has efficient mass transport and minimized shear stress can enhance cell culture efficiency. In this study, we developed a novel hydrogel tube microbioreactor using collagen proteins (ColTubes) to test this hypothesis. First, we designed an innovative micro-extruder for fabricating ColTubes loaded with cells. Our results show that collagen proteins form a dense and robust nanofiber network capable of shielding cells from hydrodynamic stress while maintaining cell mass below 400µm in diameter. The tube shell contains abundant nanopores that allow the cell culture medium to permeate and nourish the cells. Additionally, the collagen fibers serve as a substrate for cell adhesion. We show that ColTubes support high cell viability, rapid expansion, and impressive volumetric yields, offering substantial improvements over current methods. To our knowledge, ColTubes is a novel approach that has not been previously reported for cell manufacturing. ColTubes represents a scalable, cost-effective, and efficient solution for large-scale cell production.

A central hurdle in spinal cord injury (SCI) therapy involves not only reconstructing neural pathways but also overcoming the detrimental inflammatory milieu. Inspired by olfactory microenvironmental niches, we implemented a niche-targeted strategy. Our investigation defines the biological properties of ectodermal olfactory mesenchymal stem cells (OMSCs) and further elucidates their niche-targeted paracrine effect in vivo and in vitro. The findings demonstrate that OMSC-conditioned medium (OMSC-CM) delivered in fibrin hydrogel mediates potent SCI repair by concurrently protecting neurons, enhancing axonal regeneration, and suppressing destructive inflammation via IL-10 signaling. Critically, persistence of IL-10 signaling in vivo is sustained both by direct supply from OMSC-CM and by OMSC-CM-induced activation of CD206 + macrophages IL-10 . Successful neural circuit reconstruction with OMSC-CM depends on maximizing neuronal involvement in neural pathway formation.These findings may establish a special conceptual framework for developing regenerative medicine strategies in the future.

Current organoid culture systems face critical limitations: standardized growth factor formulations fail to capture patient-specific signaling requirements, while single-cell-type approaches overlook tumor-stromal interactions essential for understanding immunotherapy resistance. To address these challenges, we developed an automated biofabrication platform that systematically integrates patient-derived three-dimensional (3D) cultures with comprehensive growth factor profiling across 128 combinations. Through rigorous optimization of Matrigel concentration and gelation kinetics, we established standardized conditions achieving uniform signal distribution and quantitative reproducibility. Screening of 23 ovarian cancer patient samples identified universal growth factor combinations that consistently promoted robust cell growth while preserving parental tumor characteristics. Integration of growth factor response profiles with multi-scale genomic analysis revealed two estradiol-responsive cellular populations coordinating immunosuppression: a malignant cell fraction (MAL.PDCD5) that suppresses immune infiltration and a cancer-associated fibroblast fraction (FB.TNFSF10) that promotes immune exclusion through enhanced TGF-βsignaling. Spatial transcriptomic validation demonstrated striking mutual exclusivity between FB.TNFSF10 cells and T/NK cells in native tissue architecture. Most significantly, FB.TNFSF10 abundance emerged as a robust predictor of immune checkpoint inhibitor therapy resistance across multiple cancer cohorts, independent of conventional biomarkers. This biofabrication platform provides a scalable, reproducible framework with broad applicability beyond oncology. The systematic optimization methodology is readily adaptable to other tissue types, disease models, and high-throughput drug screening applications, representing a significant advancement in functional tissue engineering for precision medicine.

A comprehensive understanding of the female reproductive system is essential for safeguarding fertility and preventing diseases related to women's health. Organoid/organ-on-a-chip, as a promising platform, could simulate complex physiological and pathological conditions, has revolutionized our understanding and management of female reproductive health. This technology is anticipated to advance the development of more effective assisted reproductive techniques, treatments and drug screening methods. This review focuses on various organoid/organ-on-a-chip models designed to mimic the ovary, uterus, mammary gland, and vagina. Then, we introduce the current state of organoid/organ-on-a-chip in female reproductive health and highlight how these models contribute to the study of female reproductive diseases. Additionally, we discuss the limitations of organoid/organ-on-a-chip technology as well as its challenges and perspectives. Collectively, we believe that as organoid/organ-on-a-chip technology continues to evolve, it holds great potential for transforming the diagnosis and treatment of female reproductive disorders, thereby enhancing women's overall health and well-being worldwide.

Diabetes, including all forms, remains a critical global health issue, affecting over 589 million adults and causing approximately 3.4 million deaths annually. Developing more relevant in vitro models for pancreatic islet functions is crucial for advancing diabetes research and therapy. Microfluidic platforms enable precise control over experimental conditions, notably the mechanical cues within the tissue microenvironment, thereby offering a powerful tool for studying cell behavior under physiologically relevant conditions. In this study, we introduce an automated stimulation platform for single-islet glucose-stimulated insulin secretion, while insulin quantification remains off-chip. This platform incorporates an integrated micro-pump and automated fluid handling, obviating the need for external injection devices. Using both EndoC-βH5® spheroids and human donor islets, we demonstrate that the platform ensures high islet viability and functionality. This scalable and reproducible system represents a significant advancement in-depth studies of islet function, with broad applications for diabetes research and personalized treatment strategies.

Triple-negative breast cancer (TNBC) is an aggressive subtype with limited treatment options. TAS-115, a multi-receptor tyrosine kinase inhibitor, has not previously been evaluated in TNBC. Here, we investigated its therapeutic effects alone and in combination with doxorubicin (DOXO), using three-dimensional heterotypic spheroid models, including free-standing, bioprinted static, and perfused systems. TAS-115 significantly reduced cell proliferation and viability, enhanced apoptosis, and suppressed c-mesenchymal-epithelial transition/hepatocyte growth factor and PI3K/Akt/mTOR signaling. Combined treatment with DOXO further amplified these effects. In perfused bioprinted models, TAS-115 markedly inhibited tumor cell migration, highlighting its potential to limit metastatic behavior. These findings identify TAS-115 as a promising therapeutic strategy for TNBC, either as a monotherapy or in combination with chemotherapy.

3D bioprinting enables rapid fabrication of complex biological structures for tissue engineering applications. However, optimizing bioink formulation remains challenging due to complex relationships among material properties, printability, and cell viability. While the perimeter ratio (Pr) is commonly used to evaluate printability, it cannot adequately capture the full geometric fidelity required for comprehensive printability assessments, limiting robust bioink design. To address this limitation, a novel Hausdorff distance (HD) metric is employed to quantify printability, directly measuring the maximum deviation between the designed and printed structures. Furthermore, multiple machine-learning approaches were applied to alginate-hyaluronic acid (ALG-HA) composite inks and rat pheochromocytoma-derived PC12 cells to assess printability and cell viability. Rheological parameters were characterized using support vector regression (SVR) with R² ≥ 0.974. Multi-layer perceptron (MLP) regressors achieved R² values of 0.932 and 0.945 when predicting HD values of printed grid structures and cell viability, respectively. A regression-based convolutional neural network (CNN) was developed to predict HD values directly from grid structure images, achieving an R² of 0.986. Through optimization, optimal as-extruded cell viability (≥ 95%) can be achieved while maintaining high printability (HD ≤ 0.20). The optimal ink composition was further verified with good long-term cell viability and proliferation potential. This proposed AI-integrated approach can dramatically reduce ink optimization time by rapidly predicting rheological properties, printability, and cell viability from minimal experimental data.