Tissue engineering. Part C, Methods最新文献

Glioblastoma (GBM) is one of the most common malignant brain tumors, with patient mortality driven by invasion into the surrounding brain microenvironment and drug resistance. Multicellular spheroids are an increasingly common model to study GBM invasion and drug response in engineered biomaterials. However, a key design feature of tumor spheroid studies is the size of each spheroid (number of cells, diameter). Given the heterogeneous growth of GBM cells at the surgical margin, spheroids of different sizes may also have clinical relevance. Here, we define shifts in behavior and drug response of wild-type (WT) and temozolomide (TMZ)-resistant GBM spheroids as a function of initial spheroid size. GBM spheroids ranging from 1,000 to 10,000 cells in size were embedded into a methacrylamide-functionalized gelatin hydrogel. GBM spheroid size had an inverse relationship with the number of apoptotic cells. We observed significant spheroid-size-dependent effects on TMZ efficacy for both TMZ-resistant and WT cells. Interestingly, high single doses of TMZ were more effective in reducing three-dimensional migration from smaller spheroids than metronomic dosing, while high single dose and metronomic dosing were equally effective in reducing invasion for large TMZ-resistant spheroids. Our study highlights the importance of considering and reporting spheroid size for cancer tissue engineering studies considering invasion and drug resistance. It also informs future studies of residual GBM at the tumor margins most responsible for patient relapse and mortality.

In this study, we demonstrate an automated approach to efficiently and reproducibly manufacture perforated poly(ε-caprolactone) (PCL) solution electrospun tubular meshes designed for critically-sized bone defect repair. The workflow improves reproducibility and reduces fabrication time by 67% (8.7 vs. 2.7 h per 10 meshes). By directly electrospinning PCL onto a rotating cylindrical mandrel, seam-related discontinuities are eliminated, and subsequent use of an automated soldering iron system enables precise 1 mm perforations that promote vascular ingrowth during bone healing. Despite the decrease in mass of the new design compared with the original design (18.24 ± 1.5 mg for old vs. 11.48 ± 1.2 mg for new design), mechanical testing revealed similar resistance to lateral compression compared with semimanually assembled meshes. This is important to prevent collapse during surgical placement and injection of osteoinductive treatments. Further, eliminating surgical glue improves the manufacturing simplicity and scaffold reproducibility. Following implantation with bone morphogenic protein-2 loaded alginate, the new design performed similarly to the original: in vivo microcomputed tomography confirmed bone formation that significantly increased (p ≤ 0.05) over 8-weeks in an established rat femoral defect model. This study provides a novel production method of tubular scaffolds with variable dimensions and flexible perforation patterns and demonstrates improvements in fabrication efficiencies and reproducibility.

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 3D printing with a polydimethylsiloxane intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, and 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-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 for diverse tissue engineering applications.

Conventional drug screening models face a series of challenges in guiding individualized cancer treatment, including time-consuming processes, limited drug coverage, and insufficient accuracy in efficacy evaluation. This study aims to establish a convenient, rapid, and reliable drug screening protocol for evaluating individualized efficacy of chemotherapy and immunotherapy. We established an ex vivo mini-tumor culture platform by culturing tumor fragments in an air-liquid interface system, which was capable of sustaining tumor growth for at least 2 weeks and maintaining immune cell infiltration for over 1 week. Using this mini-tumor culture platform, we can evaluate the individualized therapeutic responses of different tumors to chemotherapy and immunotherapy, including gemcitabine, 5-fluorouracil, cisplatin, αPD-1 and αPD-L1. Furthermore, using this drug evaluation platform, we revealed distinct therapeutic responses to immunotherapy between immune-cold tumors and immune-hot tumors, and demonstrated the important role of the immunologic adjuvant resiquimod (R848) in enhancing immunotherapy efficacy. This mini-tumor culture protocol provides a feasible implementation approach for ex vivo personalized drug testing.

Patients with breast or prostate cancer have a high chance of developing bone metastasis, which is associated with many skeletal-related events. The development of novel bone metastasis treatments is lagging behind due to the lack of reliable models. We aimed to develop a humanized bone metastasis model comprising vital human bone discs and human metastatic cancer cells (bone metastasis discs), which were subsequently cultured ex vivo or subcutaneously implanted into nude mice. Ex vivo culture experiments confirmed that cells within the bone metastasis discs remained metabolically active, while the presence of metastatic cancer cells could be monitored using bioluminescence. Although histological analyses confirmed the presence of relevant bone cells in the human bone tissue, no apparent formation of metastatic lesions was detected over the 2-week ex vivo culture period. In contrast, subcutaneously implanted bone metastasis discs demonstrated clear metastatic lesion formation, with osteolytic characteristics, that progressed from 3 to 6 weeks after implantation for both breast and prostate cancer bone metastasis discs. Histologically, healthy bone tissue with bone marrow compartments as well as anastomosis was observed. Cisplatin treatment of ex vivo cultured bone metastasis discs significantly decreased the bioluminescent signal from (prostate) cancer cells, while no effects of cisplatin treatment were observed for in vivo implanted bone metastasis discs. Our data provide a proof of concept for an ex vivo/in vivo bone metastasis model with vital human bone and human metastatic cancer cells but require further fine-tuning to improve robustness, relevance, and quantification methods. Future research could potentially use these models for the evaluation of novel bone metastasis treatments, accelerating their potential clinical application.

During development and regeneration, bone is formed by endochondral ossification (EO) through the remodeling of a cartilage template. This complex process involves multiple cell types and interactions that cannot currently be modeled in vitro. This study aimed to develop a novel tissue-engineered human in vitro model of certain aspects of the early stages of EO by integrating cartilage which undergoes mineralization, self-assembled vascular networks, and osteoclasts into a single system. We first studied the dynamics of osteoclastogenesis and vascularization in an in vivo model of stromal cell-mediated EO, to inform our in vitro system. Next, we aimed to develop a fully human cell-based three-dimensional model of EO by combining pediatric bone marrow stromal cells differentiating into chondrocytes, osteoclasts derived from human CD14+ monocytes, and human umbilical vein endothelial cells and adipose-derived stromal cells as vessel-forming cells. We investigated how mineralizing cartilage affects osteoclast and vessel formation in vitro through separate cartilage-osteoclasts and cartilage-vessels cocultures. Finally, we combined these elements and established a complex in vitro model that supports the functionality of all these cell types and recapitulates chondrogenesis, cartilage mineralization, vessel formation and osteoclastogenesis. This integrated approach reaches unprecedented complexity and will enable new tissue engineering strategies to model skeletal diseases or cancer metastasis to the bone.

Colorectal organoids, which accurately replicate the structure and function of the human colorectal epithelium, have become a valuable platform in a broad spectrum of fundamental biological research and clinical applications. This study employs bibliometric analysis to develop a knowledge domain map specifically focusing on colorectal organoid research. Articles were sourced from the Web of Science Core Collection, and CiteSpace 6.3.R1 was utilized to analyze the literature, including outputs, journals, countries, institutions, authors, cocited authors, references, co-occurring terms, and burst terms. Subsequently, we examined prevailing research themes and focal points and identified potential future research directions within this domain. Between 2010 and 2025, a total of 719 articles related to colorectal organoid research were published. Among these, the journal Nature Communications published the highest number of papers. The United States and Utrecht University were identified as the most prolific country and institution, respectively. Hans Clevers emerged as the most prolific author, while Toshiro Sato had the highest number of cocitations, indicating that both are ideal candidates for academic collaboration. The research focus on colorectal organoids has evolved from basic biological characteristics to disease modeling and clinical applications, and further towards an in-depth exploration of functional mechanisms and precision medicines. The terms "patient-derived organoids", "disease modeling", "epithelial barrier", and "personalized medicine" have garnered significant attention between 2020 and 2025, highlighting them as promising areas for future research. Research on colorectal organoids has achieved substantial progress, positioning itself as a vital interdisciplinary field that integrates fundamental biology with clinical medicine. Future studies should focus on optimizing organoid culture methodologies, exploring functional mechanisms, and expanding clinical applications-especially in disease modeling and personalized medicine.

Evaluating the complex, three-dimensional (3D) architecture of de novo angiogenesis in artificially engineered tissue remains a significant challenge, as conventional methods like 2D histology and microimaging techniques are limited. For axial vascularization techniques, a reproducible method for complete visualization of the microcirculatory system is needed. We present an integrated workflow for high-resolution 3D visualization of neovascularization within arteriovenous (AV) loop-based tissue constructs in a rat model. An intravascular perfusion with a cationic near-infrared fluorescent dye, MHI148-polyethylenimine, was used to 3D label the patent vasculature. Following perfusion-fixation and explantation, the construct was rendered optically transparent using an ethyl cinnamate-based clearing protocol. The fluorescent signal was then imaged using confocal and light-sheet fluorescence microscopy at 7 and 28 days postimplantation. Our workflow successfully achieved high-contrast, 3D visualization of the microvascular network, allowing for whole-mount and segmental analysis of the vascular tree. At day 7, imaging delineated solely the AV loop axis while by day 28, a dense and complex, interconnected capillary plexus from the central axis demonstrated a progressive neovascularization. Downstream processing compatibility was confirmed through successful rehydration and 3D nuclear counterstaining. This workflow offers a powerful and reproducible method for detailed structural assessment of microvascular networks in large engineered constructs, overcoming key limitations of existing techniques.

Adipose tissue is an abundant and clinically accessible source of stromal cells. Stromal vascular fraction (SVF) and nanofat have been widely investigated for their regenerative potential; however, commercial systems vary considerably in yield, viability, and regulatory oversight. Most devices report fresh results only, with limited validation following cryopreservation. Mesenchymal stromal cells derived from adipose tissue have also attracted attention due to their accessibility, immunomodulatory effects, and multipotent differentiation capacity. Uvence has developed a proprietary workflow for adipose tissue processing that integrates washing, cryopreservation, thawing, and emulsification within a Human Tissue Authority-regulated laboratory. The process includes Good Manufacturing Practices (GMP) Annex 1-aligned environmental monitoring and independent quality control (QC) testing. Critically, this workflow validates postthaw cell viability, addressing a gap in current SVF/nanofat approaches. Three cryopreserved donor samples demonstrated a mean postthaw viability of ∼91% (range 90.5-92%), consistently exceeding the International Federation for Adipose Therapeutics and Science (IFATS)/ International Society for Cell and Gene Therapy (ISCT) 70% threshold. Benchmarking against global systems showed Uvence postthaw viability to be equivalent to or higher than fresh outcomes reported for enzymatic platforms (Celution, 85-91%; InGeneron, 86%) and mechanical platforms (Lipocube, Tulip, ∼96%). Unlike competitor devices, Uvence has validated freeze-thaw performance, providing a stable and compliant platform. This study also presents in vitro culture and characterization of stromal cells expanded from Uvence nanofat-derived SVF samples, including flow cytometry, morphology, and trilineage differentiation. Flow cytometry confirmed high expression of CD73, CD90, and CD105, with minimal expression of CD34/CD45, consistent with the ISCT criteria. While these findings are limited to research characterization and do not constitute approval for therapeutic use, they demonstrate that the Uvence workflow delivers a quality-focused approach to adipose tissue processing.

Background & objective: Endoscopic Submucosal Dissection (ESD) effectively treats early gastric cancer, but postoperative complications limit its clinical use. Therefore, this study examines how esophageal mucosal wound protective gels improve wound healing and reduce post-ESD complications.

Methods: The gels were characterized for physical properties and stability using rheological behavior, injectability, swelling capacity, and enzymatic degradation resistance. Biocompatibility was assessed via hemolysis testing, cytotoxicity assays, and oral mucosal irritation tests. Furthermore, wound repair potential was evaluated through cell proliferation, migration, and cell cycle analysis in Het-1A cells. Finally, in vivo recovery experiments were conducted to assess post-ESD wound healing efficacy.

Results: The gels exhibited favorable physical properties, chemical stability, and biocompatibility. Specifically, they maintained stability in the digestive tract, underwent rapid gelation at 37°C, and promoted cell proliferation. Post-ESD evaluation further revealed improved mucosal healing with no significant bleeding events.

Conclusion: The developed esophageal mucosal wound-protective gels fulfill the requirements for submucosal interventions and show promising potential for ESD wound repair via rapid in situ gelation. This platform could be adapted for various endoscopic procedures and provides new insights for digestive tract tissue engineering applications.