Biofabrication最新文献

The 3D hydrogel-based tumor model demonstrates significant potential in replicating the physiological characteristics ofin vivotumor environments for mechanistic studies and drug testing. However, the challenge persists in accurately mimicking a vascularized microtumor with a compartmentalized structure in a controlled, heterogeneous, and high-throughput manner. This study introduces a vascularized 3D tumor model that incorporates an endothelial cell (EC) barrier, created by encapsulating glioma cells and human umbilical vein endothelial cells (HUVECs) within the core (6% gelatin) and shell (10% GelMa) of core-shell microbeads, respectively. Upon culture, the tumor cells develop spheroids within the liquid core, while the HUVECs in the shell migrate and adhere to the GelMa surface, ultimately forming an EC barrier. This 3D microengineered tumor model exhibits angiogenesis in solid tumor spheroids, effectively mirroring thein vivostructure and providing relevant biochemical and biophysical properties. Notably, in comparison to 2D cell cultures, the vascularized tumor model shows significantly higher half-maximal inhibitory concentrations for the anticancer drug doxorubicin. Collectively, these findings highlight the considerable potential of engineered 3D tumor models in drug testing.

Currently, type 1 diabetes (T1D) can be treated through implantation of allogenic islets, which replenish the beta cell population, however this method requires an extensive post-implantation immunosuppressant regimen. Personalized cellular therapy can address this through implantation of an autologous cell population, induced pluripotent stem cells (iPSCs). Cellular therapy, however, requires an encapsulation device for implantation, and so to achieve this uniformly with cells in a clinical setting, bioprinting is a useful option. Bioprinting is dependent on having a bioink that is printable, retains structural fidelity after printing, and is supportive of cell type and function. While bioprinting of pancreatic islets has been demonstrated previously, success in maintaining islet function post-printing has been varied. The objective of this study is to investigate the feasibility of printing functional islets by determining the appropriate combination of bioink, printing parameters, and cell configuration. Here, we detail the successful bioprinting of both primary human islets and iPSC-derived islets embedded in an alginate/methylcellulose bioink, with functionality sustained within the construct for both cell lineages. Sc-RNAseq analysis also revealed that printing did not adversely affect the genetic expression and metabolic functionality of the iPSC-derived islets. Importantly, the iPSC-derived islets displayed comparable functionality to the primary islets, indicating the potential to act as a cell source alternative for T1D implantation.

The reconstruction of human tubular structures-characterized by adjustable small diameters (<6 mm), multifurcated morphologies, and biomimetic functionality-remains a significant challenge, particularly for researchers lacking specialized fabrication skills. In this work, we present a simple and effective strategy to fabricate freestanding, multifurcated hydrogel microtubes with tunable diameters, perfusability, and endothelialization capability by integrating stimuli-responsive hydrogels with a bubble casting technique. Leveraging the adhesive interaction between hydrogels and silicone molds, this method enables the formation of multifurcated hydrogel microtubes with uniform thickness and interconnected structures within modularly assembled molds. The integration of temperature-sensitive gelatine and photo-crosslinkable methacrylated gelatin (GelMA) permits the rapid and irreversible formation of robust hydrogel microtubes. A wide range of 2D structures including straight, L-shaped, T-shaped, bifurcated, and trifurcated microtubes can be readily produced, and further assembled into interconnected 3D microtube network using Lego-like assembly with the assistance of T- or Y-shaped adhesive connectors. The experimental results prove that the fabricated microtubes exhibit favorable physiological stability, mechanical strength, semi-permeability, hemocompatibility, cytocompatibility and anti-thrombogenicity. Moreover, the successful perfusion of whole rabbit blood and endothelialization with human umbilical vein endothelial cells (HUVECs) demonstrate their functional potential as biomimetic vascular scaffolds. Overall, our work introduces a robust, accessible, and modular strategy for generating multifurcated hydrogel microtubes featuring adjustable fine diameters. The technique is particularly suited for applications in tissue engineering and vascular modeling, and can be easily adopted by researchers across disciplines without the need for specialized equipment or training.

Existing animal and human cell models have limitations in terms of heterogeneous differences or difficulties in sufficiently reproducing arterial structures and complex cell-cell interactions. The discovery of exosome-derived biomarkers using a three-dimensional (3D) bioprinted atherosclerosis model provides a noninvasive and stable detection method and is expected to contribute to the development of early diagnosis and personalized treatment. To contribute to the discovery of exosome-derived biomarkers related to the early diagnosis and prognosis of cardiovascular diseases using a 3D bioprinted atherosclerosis model, we reproduced an arterial environment using 3D bioprinting composed of a biocompatible extracellular matrix (bioink) and various human cellsin vitro. The 3D bioprinted atherosclerosis model composed of inflammatory macrophages, coronary artery smooth muscle cells, coronary artery endothelial cells, and collagen methacryloyl (ColMA) hydrogel was treated with low-density lipoproteins to induce atherosclerosis, and the atherosclerosis model was classified into Baseline, early atherosclerosis (EA; Early Athero), and late atherosclerosis (LA; Late Athero) groups. The secreted exosomes were isolated according to the time period, and a characterization analysis was conducted to confirm the purity of the isolated exosomes. We evaluated the isolated exosomes qualitatively and quantitatively. Isolated exosomes were analyzed using proteomics and microRNA (miRNA) sequencing to verify whether the bioprinted atherosclerosis model induced atherosclerosis, and a novel EA biomarker, SERPINA11, was discovered. In conclusion, we verified that the bioprinted atherosclerosis model induced atherosclerosis and that the novel biomarker set of exosomal miRNAs (hsa-miR-143-5p and hsa-miR-6879-5p) expressed in EA and proteins (SERPINA11, AHSG, and F2) might be clinically useful in early diagnosis and prognosis.

Functional repair of full-thickness defects in the weight-bearing articular cartilage has been one of the major challenges in orthopeadics. Whereas the advanced 3D printing technique allows the construction of bionic bioscaffolds that supportin-situtissue regeneration. Herein, we developed a sort of lineage-specific biphasic scaffolds for osteochondral regeneration, fabricated via consecutive 3D-printing and lyophilization. To facilitate osteogenesis and bone formation, a porous scaffold was 3D-printed fabricated using a composite ink consisting of gelatin methacrylate (GelMA) and hydroxyapatite (HAP). To synergistically stimulate chondrogenesis and hyaline cartilage regeneration, collagen was infused into the top layers of the 3D-printed GelMA/HAP construct.In vitroculture of bone marrow mesenchymal stem cells (BMSCs) showed that the top collagen layer preferentially promoted BMSCs chondrogenic differentiation, while the GelMA/HAP composite mostly contributed to their osteogenic differentiation. This customized biphasic scaffold was then examined within the defected weight-bearing regions of full-thickness articular cartilage in rabbits, in which neocartilage, bone formation and remodeling were identified at six and twelve weeks post-implantation. Consistently to thein vitrofindings, the bottom GelMA/HAP scaffold facilitated bone formation, while the top-layer with preloaded collagen markedly augmented hyaline cartilage formationin vivo. Furthermore, it was evident that the biphasic scaffolds effectively modulated bone remodeling dynamics via inhibiting hyperactive osteoclast activities. Considering that such combinatorial biphasic scaffolds have been easily prepared and successfully utilized for cartilage defect repair, this cell-free tissue-engineered strategy holds great promise in future clinical translation.

Spheroids have become a de facto model three-dimensional tissue for studying various biological phenomena. While the technology to produce spheroids has become highly accessible and is routinely used by researchers, it has quite a long history, going through successive advances incorporating various scientific and engineering principles to acquire efficiency, accuracy, and high-throughput capability. More recently, the spheroid technology is advancing towards recapitulating complex physiological features, especially introducing extracellular components via biomaterials to more accurately portray tissue microenvironment. This review introduces and chronicles the advancement in spheroid technology in historical perspective, highlighting the key attributes of various techniques with notable examples. The spheroid technology is for convenience divided into three different generations, based on the era and the level of technological sophistication.

Diabetic wounds represent a longstanding global health challenge attributable to tissue hypoxia resulting from impaired microcirculation, which impedes crucial physiological processes essential for wound healing, such as cell proliferation and migration. Oxygen-releasing biomaterials present a novel avenue for tissue reoxygenation therapy, offering advantages over conventional hyperbaric oxygen therapy. Herein, we developed a microcosmic oxygen-releasing platform (MORP) named photosynthetic egg by utilizing egg white hydrogel with inherent bioactive factors for regenerative strength and electrostatic adsorbedChlorellabringing photosynthetic oxygen production. The dissolved oxygen concentration leaped to more than 10 mg l^-1under hypoxic conditions through manipulating supplemental dosage and illumination intensity demonstrating high flexibility and controllability of MORP.In vitroexperiments, coupled with transcriptome sequencing and quantitative real-time polymerase chain reaction analysis, demonstrated that MORP significantly augmented cell proliferation, migration, and angiogenesis, serving as a rejuvenating agent to alleviate DNA damage and cellular dysfunction in hypoxic environments. Furtherin vivoinvestigations substantiated that MORP expedited diabetic wound healing by fostering tissue regeneration, collagen deposition, and angiogenesis owing to its bioactive constituents and reoxygenation capabilities. These findings underscore the potential therapeutic efficacy of MORP as an innovative approach for managing diabetic wounds.

Bioprinting is a promising family of processes combining 3D printing with life sciences, offering the potential to significantly advance various applications. Despite numerous research efforts aimed at enhancing process modeling, optimizing capabilities, and exploring new conditions, there remains a critical need to enhance process efficiency. Experimental data are paramount for improving models. Nevertheless, it is practically unfeasible to explore a multitude of conditions (e.g. different material formulations, process parameters, machines, setups), especially given the experimental constraints of budget and time. Leveraged by in-situ bioprinting monitoring, this paper explores a set of transfer learning (TL) methods designed for resource-efficient bioprinting modeling, aiming to merge established knowledge with new experimental conditions. TL encompasses machine learning strategies focused on transferring knowledge across distinct, yet similar, domains. TL is applied to an extrusion-based bioprinting case study for printability response modeling. The knowledge acquired from a model trained on one material (the source) is transferred to a new material (the target), under conditions of limited experimental data availability. Eventually, the accuracy of the transferred model is assessed and compared against a reference no-transfer scenario, which is developed from scratch following conventional practices. Furthermore, giving high importance to the experimental effort reduction, a sensitivity analysis altering the number of experimental training points is performed to assess performances and limitations of the method. This method demonstrates the feasibility of knowledge transfer in bioprinting as a catalyst for more sophisticated applications across diverse printing conditions, materials, and technologies to advancing this technology towards achieving its full potential.

Intervertebral disc (IVD) degeneration (IVDD) is a major contributor to chronic low back pain, representing a substantial burden on the spinal healthcare system and serving as a leading cause of long-term disability worldwide. Biomimeticin vitromodels that accurately replicate histological characteristics, three-dimensional structures, and multicellular interactions are lacking. Consequently, monocultures of cell lines and two-dimensional culture models are still used to study the pathomechanisms of IVDD. We established functional multicellular IVD spheroid cultures using primary human annulus fibrosus and nucleus pulposus cells. The spheroids maintained the IVD-specific phenotype, including hypoxic conditions and lamellar structures. Additionally, the spheroid markedly increased the expression level of inflammatory mediators and chemokines in the presence of the pro-inflammatory cytokine IL-1β, a master regulator of IVDD. Furthermore, we implemented our microfluidic chemotaxis platform to investigate microglial neuroinflammation in response to our reconstituted IVD spheroid models. Transcriptome sequencing revealed that microglia stimulated by potential contributing factors derived from IVDD spheroids exhibited a significant upregulation of the expression levels of chemotactic factors and cytokines including CCL-2, -3, -4, -5, IL-8 and IL-6 (p< 0.05). Moreover, we observed considerable activation and infiltration of microglia induced by soluble factors derived from IVDD spheroids, which are expected to occur during IVDD. The chemotactic effects on microglia were reduced upon the neutralization of CCL-2 or IL-8 or inhibition of NF-κB signaling. These robustin vitroIVD spheroids can be used to model IVDD and provide a valuable platform for the assessment and development of IVDD therapeutics.

During brain metastasis, tumor cells interact with the surrounding stroma, including neurons and astrocytes, to create a tumor-promoting microenvironment. However, the molecular and cellular factors driving tumor-neural stroma interactions remain unclear. Here, we developed a co-culture model of metastatic melanoma by combining metastatic melanoma cells with dissociated human iPSC-derived cerebral organoids, consisting of neurons and astrocytes, in a microfluidic device. We cultured these astrocytes and neurons in a 3D hydrogel that contained a domain with metastatic melanoma cells. This approach generated a spatially organized co-culture system with no physical boundary between the tumor and stromal compartments. Then, we leveraged several imaging modalities to study tumor-stroma interactions and changes in the microenvironment. Using non-destructive, luminescence-based methods, we spatially resolved changes in cell viability, metabolite concentration, and other biochemical parameters. We also used luminescence to analyze the effect of radionuclides on tumor cell viability and used PET imaging to monitor their diffusion across the system.