Biofabrication最新文献

Hydrogels are widely used in tissue engineering, but conventional homogeneous polymerization often creates dense matrices that hinder cell migration and restrict extracellular matrix production. The motivation of this project was to overcome these limitations by developing a heterogeneously crosslinkable hydrogel platform that enables both cell migration and matrix deposition. We present a two-step heterogeneous polymerization approach that introduces spatial variations in matrix density, producing tunable, cell-sized pores that promote migration, proliferation, and matrix synthesis. As an implementation, gelatin was pre-assembled into microribbon-like building blocks using a dynamic molding process, methacrylated to introduce crosslinkable groups, chemically modified, washed, and freeze-dried. Upon rehydration, the ribbons formed a moldable paste that could be mixed with cells and photo-crosslinked into scaffolds within situ-formed, cell-sized pores. The main novelty of this method is the introduction of chemical modifications with methacrylic anhydride (MAA), acetic anhydride (AceA), and succinic anhydride (SucA), which enable a controlled two-step heterogeneous polymerization and allow independent tuning of scaffold microstructure, mechanics, and degradation. AceA reduced crosslink density and accelerated degradation, whereas SucA promoted swelling, enhanced mechanical strength, and slowed degradation. Cell studies revealed that SucA-modified scaffolds supported superior adhesion and proliferation compared to AceA-modified and unmodified controls. Such work may significantly impact the design of next-generation scaffolds by providing a versatile platform that integrates structural, mechanical, and biochemical control for regenerative medicine applications.

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.

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.

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.

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.

The transplantation of human bone marrow mesenchymal stem cells (hMSCs) exhibits promising therapeutic effects in the treatment of myocardial infarction (MI), however, its clinical application is limited due to the low survival rate of the transplanted cells. Three-dimensional (3D) bioprinted tissue engineering patches have demonstrated efficacy as a delivery approach to enhance the viability and engraftment of stem cells. In this study, we have developed a novel hMSCs tissue-engineered patch equipped with a nano-slow-release system using 3D bioprinting technology. The patch is based on a matrix material consisting of methacrylated gelatin (GelMA) and chitosan nanoparticles loaded with vascular endothelial growth factor (VEGF), which possesses pro-angiogenic effects. The resulting patch demonstrated excellent compatibility with hMSCs and enabled stable, sustained VEGF release.In vivoresults showed that the patch significantly reduced cardiomyocyte apoptosis three days after MI, and improved cardiac function and myocardial fibrosis at 28 d post-surgery. These effects were closely associated with the patch's potent angiogenic properties and favorable stem cell survival. In conclusion, this study successfully developed a 3D-printed tissue engineering patch with strong potential for clinical application, offering a promising new approach for the treatment of MI.

Bottom-up tissue engineering has gained significant interest for its ability to recreate the complexity of human organs by assembling functional tissue units through techniques such as extrusion-based bioprinting (EBB). To enable the future biofabrication of human-scale organs, new bioinks for EBB must be developed that facilitate the formation of a functional vascular network within the biomaterial. Without a vascular system, high cell densities within the construct struggle to survive due to the diffusion limits of oxygen and nutrients. Additionally, the bioink must exhibit sufficient printability to accurately recreate the 3D CAD model. In the current work, elastin is modified with norbornene groups to enable step-growth polymerization with thiolated gelatin, resulting in a novel hybrid biomaterial. Unmodified gelatin and porogens are incorporated into the elastin-gelatin hydrogel to enhance printability in EBB and increase porosity, respectively. When only unmodified gelatin is added to the elastin-gelatin hydrogel, shape fidelity on a continuous platform is excellent, and the bioink successfully bridges gaps up to 8 mm with a 100% success rate. Upon addition of alginate gel porogen (AGP), quality of printing on a continuous platform is maintained, but the gap-bridging capability becomes limited to gaps smaller than 4 mm. Nonetheless, the elastin-gelatin hydrogel supplemented with both unmodified gelatin and AGP is preferred, as it promotes superior vascular development compared to a wide range of other bioinks, with vasculogenesis-driven self-assembly of embedded endothelial cells reaching a total vascular network length of 26 ± 6 mm mm^-3and angiogenic sprouting from vascularized spheroids reaching a total sprout length of 4 ± 2 mm within the hydrogel by day 7. A bioink that supports this level of vascular development while maintaining sufficient printability represents a valuable addition to the toolkit for bottom-up tissue engineering using EBB.

Liposomes, as one of the most promising and rapidly evolving drug delivery systems, are highly valued for their biocompatibility, ability to encapsulate diverse drugs, controlled release, and targeted delivery, offering enhanced therapeutic effects with reduced toxicity. However traditional methods for synthesizing liposomes still exhibit problems such as uncontrollable particle size and uneven distribution, reducing passive targeting efficiency and compromising treatments In present study, we introduce a novel alternating current electrokinetic mixing-assisted micro-synthesis method for liposome production, utilizing a novel custom (mold-extraction) approach to fabricate a 3D-structured microfluidic chip with parallel electrodes along both sides of the channel. Unlike traditional methods, where etched thin electrodes often result in non-uniform electric fields and leakage, the present method enables the placement of 3D electrodes with channel thickness, minimizes electrode distance, and allows for the generation of a strong, uniform electric field at low voltages. Consequently, controllable ultra-fast active mixing is achieved, resulting in the controlled and adjustable synthesis of liposomes with uniform size distributions. The effects of flow rate,E(electric field intensity), and frequency on the synthesis of liposomes were investigated. Additionally, studies demonstrated that drug encapsulation efficiency can be precisely controlled by modulating the applied electric field, a capability that was further validated through cellular experiments. This study presents a straightforward and adjustable approach for the precise synthesis of liposomes, which can be utilized to develop customized drug delivery systems.

The fabrication of anatomically accurate, cellularized heart valve substitutes remains a significant challenge in tissue engineering, particularly for pediatric and patient-specific applications. While three-dimensional (3D) bioprinting enables the creation of complex geometries, it often compromises cell viability and lacks the precision required for small-scale constructs. In this study, we present a high-fidelity, reproducible molding technique using 3D-printed sugar glass molds to engineer custom, alginate-based hydrogel cellularized heart valves. Human adipose-derived stromal cells (ASCs) were used as the cell source due to their accessibility and regenerative potential. This approach overcomes the limitations of conventional molding and bioprinting by enabling the reproduction of intricate anatomical features, including the sinuses of Valsalva, which are critical for physiological hemodynamics. The molding method maintains high cell viability (>90%) at the time of fabrication and the process supports both scalability and automation. Sugar glass molds for valve sizes from 16 to 26 mm inner diameter were printed with 90% of the mold surface within a ±0.3 mm deviation of the reference computer-aided design model. Cellularized valves cultured in a custom perfusion bioreactor retained structural integrity and cell viability over a 14 d period. This biofabrication strategy offers a promising platform for engineering patient-specific heart valves and also lays the groundwork forin vitrodisease modeling, including valve mineralization, using living cells such as ASCs.