Glioblastoma (GBM) remains a formidable challenge due to its aggressive nature, protected location within the brain, and resistance to conventional treatments. Its complex tumor microenvironment (TME), coupled with the blood-brain barrier (BBB), hinders drug delivery leading to poor treatment outcomes. Nanoparticles (NPs) offer a promising solution, as they can improve the pharmacokinetic properties of anticancer agents. By functionalizing NPs with targeting molecules, researchers aim to enhance drug concentration in the brain. However, developing effective NP-based therapies requires robust in vitro models that accurately capture the complexities of GBM. Two-dimensional (2D) and three-dimensional (3D) cell culture models provide a versatile platform for studying NP-cell interactions. By customizing cell types, incorporating TME components, and adjusting flow conditions, researchers can tailor these models to specific research questions. While 2D models offer a simpler starting point, 3D models, such as multicellular spheroids and organoids, can more accurately replicate the complex TME, including the BBB and tumor heterogeneity. These models enable a more comprehensive evaluation of NP efficacy and safety, ultimately accelerating drug development and reducing reliance on animal testing.

Reconstructing auricular tissue is challenging because ear cartilage has few blood vessels and limited regenerative capacity. Traditional methods that utilize autologous costal cartilage or synthetic polymers often lead to donor site morbidity and suboptimal biocompatibility. In this study, we introduce 3D-printed scaffolds composed of decellularized human cartilage-derived bioink combined with polycaprolactone (PCL), designed to enhance both tissue regeneration and mechanical stability. The decellularization process effectively removed cellular components while preserving glycosaminoglycan and total collagen, comparable to those in native cartilage. We formulated the bioink by incorporating decellularized human cartilage particles into hyaluronic acid and carboxymethyl cellulose gels, optimizing the rheological properties for 3D printing. In vitro tests demonstrated that the decellularized human cartilage-derived bioink exhibited no cytotoxicity and facilitated the migration and chondrogenic differentiation of human adipose-derived stem cells. We fabricated 3D-printed scaffolds using this bioink combined with PCL and evaluated their performance in rabbits over a one-year implantation period. Our results indicated that the scaffolds maintained structural integrity throughout the year and exhibited significant neovascularization and chondrogenesis. Histological analysis revealed increased blood vessel formation in scaffolds with higher ratios and greater decellularized cartilage content with notable differences observed across varying porosities. These findings suggest that 3D-printed scaffolds with decellularized human cartilage-derived bioink and PCL offer a promising approach for auricular reconstruction, potentially improving outcomes for patients with microtia.

The poor survival of ovarian cancer patients is linked to their high likelihood of relapse. In spite of full apparent macroscopic clearance, tumor recurrences arise from cells that are resistant to primary chemotherapy in the form of minimal residual disease (MRD). MRD exhibits distinct molecular drivers from bulk cancer and therefore necessitates alternative therapeutic strategies. However, there is a lack of 3D models that faithfully recapitulate MRD ex vivo for therapy development. This study constructs microfluidics-based 3D microtumors to generate a clinically-relevant model for ovarian cancer MRD. The microtumors recapitulate the non-genetic heterogeneity of ovarian cancer, capturing the "Oxford Classic" five molecular signatures. Gene expression in the 3D microtumors aligns closely with MRD from ovarian cancer patients and features the upregulation of fatty acid metabolism genes. Finally, the MRD 3D microtumors respond to the approved fatty acid oxidation inhibitor, perhexiline, demonstrating their utility in drug discovery. This system might be used as a drug-testing platform for the discovery of novel MRD-specific therapies in ovarian cancer.

This study aimed to examine the influence of matrix stiffness on osteogenic differentiation via epigenetic mechanisms in human periodontal ligament stem cells (hPDLSCs), with implications for understanding orthodontic tooth movement.

hPDLSCs were cultured on substrates with varying stiffness (soft and stiff). Dot blot and immunofluorescence techniques were employed to measure global DNA methylation levels. RT-qPCR and alkaline phosphatase (ALP) activity assays were conducted to assess differences in DNA methylation and osteogenic potential. Additionally, ELISA was used to quantify DNA methyltransferase content and activity.

hPDLSCs on stiffer substrates exhibited increased 5-methylcytosine (5-mC) and higher global DNA methylation levels than those on soft substrates. With increased matrix stiffness, DNMT3A and DNMT3B mRNA expression levels rose. hPDLSCs on stiff matrices also showed elevated DNMT3B enzyme content and osteogenic activity. When global DNA methylation was reduced, mRNA levels of RUNX2, ALP, and Col-1 decreased, along with a notable reduction in ALP staining intensity in the inhibitor group.

Matrix stiffness is positively associated with global DNA methylation, with DNMT3B likely mediating this regulation in hPDLSCs. Furthermore, DNA methylation levels are positively linked to the osteogenic capability of hPDLSCs.

Animal models are commonly used for drug screening before clinical trials. However, developing these models is time-consuming, and the results obtained from these models may differ from clinical outcomes due to the differences between animals and humans. To this end, 3D bioprinting offers several advantages for drug screening, such as high reproducibility and improved throughput, in addition to the human cells that can be used to generate these models. Here, we report the development of an animal patient-derived in vitro breast cancer model for drug screening using digital light processing (DLP) bioprinting. These bioprinted models demonstrated good cytocompatibility and preserved phenotypes of the cells. DLP enabled rapid fabrication with blood vessel-like channels to replicate, to a good extent, the tumor microenvironment. Our findings suggested that the improved microenvironment, provided by vascular structures within the bioprinted models, played a crucial role in reducing the chemoresistance of drugs. In addition, the correlation of the in vitro and in vivo drug-screening results was preliminarily performed to evaluate the predictive feasibility of this bioprinted model, suggesting a potential strategy for the design of future drug-testing platforms.

The use of fluorescent labels is the most common tool to visualize cells. However, the internalization of dye molecules often modifies the cell behavior. In this paper we demonstrate that it is possible to transiently label cells using a 3D scaffold, a hydrogel, covalently functionalized with luminescent cyclometalated iridium(III) complexes. The unique feature of our design is that the complexes are emissive only when they interact with the cell membrane while their emission is quenched in water. We exploited this feature to perform real-time and staining-free cell visualization and imaging. Iridium functionalized hydrogels are very weakly luminescent when immerged in culture media. When cells are added to them, they interact with the iridium complexes, covalently linked to the gel, and their lipophilic membrane "switches on" the luminescence enabling a clear and dynamic, real-time 3D visualization of cell proliferation. A complete photophysical and biological study of the materials is presented which demonstrates the potential of our methodology for 3D-realtime cell tracking.

The combination of 3D in vitro cell culture and microfluidic technology has emerged as a powerful approach in biomedical engineering. It offers a more physiologically relevant model compared to traditional 2D cell cultures by allowing the assembly of micro-sized cellular structures, known as microgels. These microgels can be prepared and fabricated to mimic the in vivo characteristics of an ECM. We report here an economical and feasible microfluidic 3D in vitro culture platform that offers real-time monitoring of cellular proliferation by encapsulating pH-sensing carbon dots (CDs) with cells in the microgels. These CDs were shown to effectively evaluate proliferation within cell-encapsulated microgels in comparison with the traditional Alamar blue assay. The biggest advantage of this platform is its ability to co-culture different cell types, achieved by encapsulating the cells within individual microgels, spatially separating them while maintaining close proximity. In this modular system, each microgel acts as a unit of a specific cell type, allowing easy retrieval of cells and control over cell densities. We established the efficacy of this concept by co-culturing Huh-7 and NIH-3T3 cells within different microgel combinations, under both static and dynamic flow conditions. The heterotypic interactions were explored by assessing the functionality using albumin assay and CYP3A4 gene expression studies, along with performing drug toxicity assays. The functionality studies confirmed results from existing literature studies by showing an improved hepatic function in the presence of NIH-3T3, even in the dynamic state. This platform can be expanded to include multiple cell types, creating a complex tissue-like effect without requiring spatial patterning techniques.

Three-dimensional (3D) disease modeling and drug screening systems have become important in tissue engineering, drug screening, and development. The newly developed systems support cell and extracellular matrix (ECM) interactions, which are necessary for the formation of the tissue or an accurate model of a disease. Hydrogels are favorable biomaterials due to their properties: biocompatibility, high swelling capacity, tunable viscosity, mechanical properties, and their ability to biomimic the structure and function of ECM. They have been used to model various diseases such as tumors, cancer diseases, neurodegenerative diseases, cardiac diseases, and cardiovascular diseases. Additive manufacturing approaches, such as 3D printing/bioprinting, stereolithography (SLA), selective laser sintering (SLS), and fused deposition modeling (FDM), enable the design of scaffolds with high precision; thus, increasing the accuracy of the disease models. In addition, the aforementioned methodologies improve the design of the hydrogel-based scaffolds, which resemble the complicated structure and intricate microenvironment of tissues or tumors, further advancing the development of therapeutic agents and strategies. Thus, 3D hydrogel-based disease models fabricated through additive manufacturing approaches provide an enhanced 3D microenvironment that empowers personalized medicine toward targeted therapeutics, in accordance with 3D drug screening platforms.

Exposure to PM2.5 is associated with neurotoxicity and mitochondrial dysfunction. Resveratrol, a natural polyphenol, has demonstrated antioxidant and neuroprotective properties. Still, its efficacy in mitigating PM2.5-induced damage in human neural stem cells (hNSCs) and within a 3D scaffold system remains underexplored.

This study investigated the protective effects of resveratrol against PM2.5-induced damage in hNSCs and within a 3D scaffold system.

Assess cell viability using MTT and LIVE/DEAD assays and measure caspase activity by fluorescence analysis. Quantify gene and protein expression of key regulatory pathways using qPCR and Western blotting. Then, mitochondrial function was analyzed by measuring ATP production, mitochondrial mass, maximal respiratory rate, COX activity, membrane potential, TEM, and immunofluorescence staining. In addition, 3D scaffolds created by the CELLINK INKREDIBLE bioprinter were used to study the effect of resveratrol on PM2.5-induced hNSCs damage.

Resveratrol significantly improved cell viability and reduced caspase-3 and caspase-9 activities in PM2.5-treated hNSCs. Resveratrol treatment upregulated TrKBR, PI3K, AKT, CREB, PPARα, PPARγ, SIRT1 and AMPK expression. It restored mitochondrial function by increasing ATP production, mitochondrial mass, maximal respiratory rate, COX activity, and membrane potential. Using a 3D scaffold demonstrated resveratrol's potential to maintain mitochondrial function and cellular health under PM2.5 exposure.

Resveratrol can effectively reduce neurotoxicity and mitochondrial dysfunction caused by PM2.5 in hNSCs. Its protective effects against PM2.5-induced toxicity in hNSCs within a 3D bioprinted model highlight this study's translational potential. These findings emphasize its potential as a therapeutic agent against environmental neurotoxins and the development of neuroprotective strategies.

Human neural stem cells (hNSCs) have the potential to differentiate into various neural cell types, including cortical neurons, which are of particular interest for understanding and treating neurodegenerative diseases. However, traditional 2D culture methods are limited in their ability to accurately mimic the physiologically relevant microenvironment, leading to slow differentiation rates and low yields of mature neurons. In this study, we developed and optimized 3D chitosan scaffolds to promote the more efficient differentiation of hNSCs into cortical neurons. These scaffolds provide a tunable, biocompatible, and mechanically favorable environment, supporting enhanced cell-to-cell interactions and mimicking the extracellular matrix more effectively than 2D systems. The differentiation process was further accelerated by preseeding scaffolds with hNSCs, leading to increased expression of key cortical neuron markers, such as MAP2 and TUBB3, within a 14-day period. Compared to Geltrex-coated controls, the preseeded scaffolds demonstrated superior cell adhesion, viability, and differentiation efficiency, with significant upregulation of mature cortical neuron markers. Our findings suggest that chitosan-based 3D culture systems represent a promising platform for improving the differentiation of hNSCs, offering a faster and more reliable method to generate cortical neurons for neurodegenerative disease research and potential therapeutic applications.

In alignment with the global movement toward reducing animal testing, several reconstructed human epidermis (RHE) models have been created for conducting skin irritation tests. These models have undergone development, verification, validation, and integration into OECD TG 439. Our team has introduced a novel in-house RHE named GB-RHE, and we adhere to OECD TG 439 to pre-validate the model and test its potential employment for nanoparticle irritation studies. GB-RHE exhibits morphological, biochemical, and physiological attributes equivalent to the human epidermis, featuring well-differentiated multilayered viable keratinocytes with a robust barrier function. The performance of the GB-RHE model was evaluated using ten reference chemicals, following the performance standard of OECD TG 439. The results demonstrated commendable predictive capacity and showed that titanium dioxide nanoparticles (TiO2 NPs) are 'non-irritant' to the human epidermis following the globally harmonized classification system. However, although the histological analysis did not show morphological changes, transmission electron micrographs demonstrated that TiO2 NPs can be internalized, reaching the external viable layers of the epidermis. This study demonstrates that in addition to the potential of the GB-RHE model to evaluate skin irritation, this model also has the potential to evaluate the skin toxicity of NPs and carry out cell internalization studies.

Bone related disorders are highly prevalent, and many of these pathologies still do not have curative and definitive treatment methods. This is due to a complex interplay of multiple factors, such as the crosstalk between different tissues and cellular components, all of which are affected by microenvironmental factors. Moreover, these bone pathologies are specific, and current treatment results vary from patient to patient owing to their intrinsic biological variability. Current approaches in drug development to deliver new drug candidates against common bone disorders, such as standard two-dimensional (2D) cell culture and animal-based studies, are now being replaced by more relevant diseases modelling, such as three-dimension (3D) cell culture and primary cells under human-focused microphysiological systems (MPS) that can resemble human physiology by mimicking 3D tissue organization and cell microenvironmental cues. In this review, various technological advancements for in vitro bone modeling are discussed, highlighting the progress in biomaterials used as extracellular matrices, stem cell biology, and primary cell culture techniques. With emphasis on examples of modeling healthy and disease-associated bone tissues, this tutorial review aims to survey current approaches of up-to-date bone-on-chips through MPS technology, with special emphasis on the scaffold and chip capabilities for mimicking the bone extracellular matrix as this is the key environment generated for cell crosstalk and interaction. The relevant bone models are studied with critical analysis of the methods employed, aiming to serve as a tool for designing new and translational approaches. Additionally, the features reported in these state-of-the-art studies will be useful for modeling bone pathophysiology, guiding future improvements in personalized bone models that can accelerate drug discovery and clinical translation.

Three-dimensional (3D) bioprinting holds immense promise for advancing stem cell research and developing novel therapeutic strategies in the field of neural tissue engineering and disease modeling. This paper critically analyzes recent breakthroughs in 3D bioprinting, specifically focusing on its application in these areas. We comprehensively explore the advantages and limitations of various 3D printing methods, the selection and formulation of bioink materials tailored for neural stem cells, and the incorporation of nanomaterials with dual functionality, enhancing the bioprinting process and promoting neurogenesis pathways. Furthermore, the paper reviews the diverse range of stem cells employed in neural bioprinting research, discussing their potential applications and associated challenges. We also introduce the emerging field of 4D bioprinting, highlighting current efforts to develop time-responsive constructs that improve the integration and functionality of bioprinted neural tissues. In short, this manuscript aims to provide a comprehensive understanding of this rapidly evolving field. It underscores the transformative potential of 3D and 4D bioprinting technologies in revolutionizing stem cell research and paving the way for novel therapeutic solutions for neurological disorders and injuries, ultimately contributing significantly to the advancement of regenerative medicine. STATEMENT OF SIGNIFICANCE: This comprehensive review critically examines the current bioprinting research landscape, highlighting efforts to overcome key limitations in printing technologies-improving cell viability post-printing, enhancing resolution, and optimizing cross-linking efficiencies. The continuous refinement of material compositions aims to control the spatiotemporal delivery of therapeutic agents, ensuring better integration of transplanted cells with host tissues. Specifically, the review focuses on groundbreaking advancements in neural tissue engineering. The development of next-generation bioinks, hydrogels, and scaffolds specifically designed for neural regeneration complexities holds the potential to revolutionize treatments for debilitating neural conditions, especially when nanotechnologies are being incorporated. This review offers the readers both a comprehensive analysis of current breakthroughs and an insightful perspective on the future trajectory of neural tissue engineering.

Oncological diseases consistently occupy leading positions among the most life-threatening diseases, including in highly developed countries. At the same time, the second most common cause of cancer death is colorectal cancer. The current level of research shows that the development of effective therapy, in this case, requires a new grade of understanding processes during the emergence and development of a tumor. In particular, the concept of cancer stem cells that ensure the survival of chemoresistant cells capable of giving rise to new tumors is becoming widespread. To provide adequate conditions that reproduce natural processes typical for tumor development, approaches based on increasingly complex cellular systems are being improved. This review discusses the main strategies that allow for the study of the properties of tumor cells with an emphasis on colorectal cancer stem cells. The features of working with tumor cells and the advantages and disadvantages of 2D and 3D culture systems are considered.

Introduction: Pancreatic cancer (PC) is one of the most aggressive and lethal malignancies, calling for enhanced research. Pancreatic ductal adenocarcinoma (PDAC) represents 70-80% of all cases and is known for its resistance to conventional therapies. Carbon-ion radiotherapy (CIRT) has emerged as a promising approach due to its ability to deliver highly localized doses and unique radiobiological properties compared to X-rays. In vitro radiobiology has relied on two-dimensional (2D) cell culture models so far; however, these are not sufficient to replicate the complexity of the in vivo tumor architecture. Three-dimensional (3D) models become a paradigm shift, surpassing the constraints of traditional models by accurately re-creating morphological, histological, and genetic characteristics as well as the interaction of tumour cells with the microenvironment. Materials and Methods: This study investigates the survival of pancreatic cancer cells in both 2D and spheroids, a 3D model, following photon, proton, and carbon-ion irradiation by means of clonogenic, MTT, spheroid growth, and vitality assays. Results: Our results demonstrate that carbon ions are more efficient in reducing cancer cell survival compared to photons and protons. In 2D cultures, carbon-ion irradiation reduced cell survival to approximately 15%, compared to 45% with photons and 30% with protons. In the 3D culture model, spheroid growth was similarly inhibited by carbon-ion irradiation; however, the overall survival rates were higher across all irradiation modalities compared to the 2D cultures. Carbon ions consistently showed the highest efficacy in reducing cell viability in both models. Conclusions: Our research highlights the pivotal role of 3D models in unraveling the complexities of pancreatic cancer radiobiology, offering new avenues for designing more effective and precise treatment protocols.

The dynamic interaction between cells and their substrate is a cornerstone of biomaterial-based tissue regeneration focused on unraveling the complex factors that govern this crucial relationship. A key challenge is translating physical cues from 2D to 3D due to limitations in current biofabrication techniques. In response, this study introduces an innovative approach that combines additive and subtractive manufacturing for precise surface patterning of 3D printed scaffolds. Using poly(𝜀-caprolactone) as the scaffold material, polymeric fibers are 3D printed and subsequently laser-engraved with femtosecond laser to precisely create controlled microtopographies, including microgrooves (10 and 80 µm in width) and micropits (25 µm in diameter). Testing shows that the process does not compromise the mechanical properties of the fibers, which is critical for structural applications in tissue engineering. Human mesenchymal stem cells are used to investigate the effects of these topographical features on cell behavior. The 10 µm wide microgrooves notably enhance cell attachment, with cells aligning in elongated forms along the grooves, while micropits and unpatterned surfaces promote polygonal cell shapes. This combined approach demonstrates that precisely engineered microtopographies on 3D printed scaffolds can better mimic the natural extracellular matrix, improving cellular responses and offering a promising strategy for advancing tissue regeneration.

Oncolytic virotherapy shows promise as a cancer treatment approach; however, its systemic application is hindered by antibody neutralization. This issue can be overcome by using mesenchymal stem cells (MSCs) as carrier cells for oncolytic viruses (OVs). However, it remains elusive whether MSC source influences the antitumor effect. Here, we demonstrate that their source affects the migration ability and oncolytic activity of OV-loaded MSCs. Among human MSCs derived from different tissues, bone marrow-derived MSCs (BMMSCs) showed a high migration ability toward cancer cells in two- and three-dimensional MSC-cancer cell co-culture models. Comprehensive gene expression and Gene Ontology-based functional analyses suggested that genes involved in cell migration and cytokine response influence the cancer-specific tropism of BMMSCs. Furthermore, MSC origin affected the susceptibility to OVs, including cytotoxicity resistance and OV release from MSCs. MSC-mediated OV delivery significantly increased the viral spread and antitumor activity compared with delivery by OVs alone, and OV-loaded BMMSCs demonstrated the most potent antitumor effect among OV-loaded MSCs. Our results offer promising insights into cancer gene therapy with carrier cells and can help with the selection of an appropriate MSC source for MSC-based OV therapy.

Monitoring cell secretion in complex microenvironments is crucial for understanding cellular behavior and advancing physiological and pathological research. While traditional cell culture methods, including organoids and spheroids, provide valuable models, real-time monitoring of cell secretion of signaling molecules remains challenging. Integrating advanced monitoring technologies into these systems often disrupts the delicate balance of the microenvironment, making it difficult to achieve sensitivity and specificity. This review explored recent strategies for integrating the monitoring of cell secretion of signaling molecules, crucial for understanding and replicating cell microenvironments, within cell culture platforms, addressing challenges such as non-adherent cell models and the focus on single-cell methodologies. We highlight advancements in biosensors, microfluidics, and three-dimensional culture methods, and discuss their potential to enhance real-time, multiplexed cell monitoring. By examining the advantages, limitations, and future prospects of these technologies, we aim to contribute to the development of integrated systems that facilitate comprehensive cell monitoring, ultimately advancing biological research and pharmaceutical development.

Since the discovery that exosomes can exchange genes, their potential use as tools for tissue regeneration, disease diagnosis, and therapeutic applications has drawn significant attention. Emerging three-dimensional (3D) printing technologies, such as bioprinting, which allows the printing of cells, proteins, DNA, and other biological materials, have demonstrated the potential to create complex body tissues or personalized 3D models. The use of 3D spheroids in bioprinting facilitates volumetric tissue reconstruction and accelerates tissue regeneration via exosome secretion. In this review, we discussed a convergence approach between two promising technologies for bioprinting and exosomes in regenerative medicine. Among the various 3D cell culture methods used for exosome production, we focused on spheroids, which are suitable for mass production by bioprinting. We then summarized the research results on cases of bioprinting applications using the spheroids and exosomes produced. If a large number of spheroids can be supplied through bioprinting, the spheroid-exosome-based bioprinting technology will provide new possibilities for application in tissue regeneration, disease diagnosis, and treatment.

Triple-negative breast cancer (TNBC), the most aggressive subtype of breast cancer lacks estrogen, progesterone, and HER2 receptors and hence, is therapeutically challenging. Towards this, we studied an alternate therapy by repurposing metformin (FDA-approved type-2 diabetic drug with anticancer properties) in a 3D-scaffold culture, with electrical pulses. 3D cell culture was used to simulate the tumor microenvironment more closely and MDA-MB-231, human TNBC cells, treated with both 5 mM metformin (Met) and 8 electrical pulses at 2500 V/cm, 10 µs (EP1) and 800 V/cm, 100 µs (EP2) at 1 Hz were studied in 3D and 2D. They were characterized using cell viability, reactive oxygen species (ROS), glucose uptake, and lactate production assays at 24 h. Cell viability, as low as 20 % was obtained with EP1 + 5 mM Met. They exhibited 1.65-fold lower cell viability than 2D with EP1 + 5 mM Met. ROS levels indicated a 2-fold increase in oxidative stress for EP1 + 5 mM Met, while the glucose uptake was limited to only 9 %. No significant change in the lactate production indicated glycolytic arrest and a non-conducive environment for MDA-MB-231 growth. Our results indicate that 3D cell culture, with a more realistic tumor environment that enhances cell death using metformin and electrical pulses could be a promising approach for TNBC therapeutic intervention studies.

Extracellular vesicles (EV) are a heterogeneous group of lipid particles excreted by cells. They play an important role in regeneration, development, inflammation, and cancer progression, together with the extracellular matrix (ECM), which they constantly interact with. In this review, we discuss direct and indirect interactions of EVs and the ECM and their impact on different physiological processes. The ECM affects the secretion of EVs, and the properties of the ECM and EVs modulate EVs' diffusion and adhesion. On the other hand, EVs can affect the ECM both directly through enzymes and indirectly through the modulation of the ECM synthesis and remodeling by cells. This review emphasizes recently discovered types of EVs bound to the ECM and isolated by enzymatic digestion, including matrix-bound nanovesicles (MBV) and tissue-derived EV (TiEV). In addition to the experimental studies, computer models of the EV-ECM-cell interactions, from all-atom models to quantitative pharmacology models aiming to improve our understanding of the interaction mechanisms, are also considered. STATEMENT OF SIGNIFICANCE: Application of extracellular vesicles in tissue engineering is an actively developing area. Vesicles not only affect cells themselves but also interact with the matrix and change it. The matrix also influences both cells and vesicles. In this review, different possible types of interactions between vesicles, matrix, and cells are discussed. Furthermore, the united EV-ECM system and its regulation through the cellular activity are presented.

An ideal extracellular matrix (ECM) replacement scaffold in a three-dimensional cell (3D) culture should induce in vivo-like interactions between the ECM and cultured cells. Highly hydrophilic polyvinyl alcohol (PVA) nanofibers disintegrate upon contact with water, resulting in the loss of their fibrous morphology in cell cultures. This can be resolved by using chemical crosslinkers and post-crosslinking. A crosslinked, water-stable, porous, and optically transparent PVA nanofibrous membrane (NM) supports the 3D growth of various cell types. The binding of cells attached to the porous PVA NM is low, resulting in the aggregation of cultured cells in prolonged cultures. PVA NMs containing integrin-binding peptides of fibronectin and laminin were produced to retain the blended peptides as cell-binding substrates. These peptide-blended PVA NMs promote peptide-specific cell adherence and growth. Various cells, including epithelial cells, cultured on these PVA NMs form layers instead of cell aggregates and spheroids, and their growth patterns are similar to those of the cells cultured on an ECM-coated PVA NM. The peptide-retained PVA NMs are non-stimulatory to dendritic cells cultured on the membranes. These peptide-retaining PVA NMs can be used as an ECM replacement matrix by providing in vivo-like interactions between the matrix and cultured cells.

Cell spheroids (esp. organoids) as 3D culture platforms are popular models for representing cell-cell and cell-extracellular matrix (ECM) interactions, bridging the gap between 2D cell cultures and natural tissues. 3D cell models with spatially organized multiple cell types are preferred for gaining comprehensive insights into tissue pathophysiology and constructing in vitro tissues and disease models because of the complexities of natural tissues. In recent years, an assembly strategy using cell spheroids (or organoids) as living building blocks has been developed to construct complex 3D tissue models with spatial organization. Here, a comprehensive overview of recent advances in multispheroid assembly studies is provided. The different mechanisms of the multispheroid assembly techniques, i.e., automated directed assembly, noncontact remote assembly, and programmed self-assembly, are introduced. The processing steps, advantages, and technical limitations of the existing methodologies are summarized. Applications of the multispheroid assembly strategies in disease modeling, drug screening, tissue engineering, and organogenesis are reviewed. Finally, this review concludes by emphasizing persistent issues and future perspectives, encouraging researchers to adopt multispheroid assembly techniques for generating advanced 3D cell models that better resemble real tissues.

The liver has excellent regeneration potential and attains complete functional recovery from partial hepatectomy. The regenerative mechanisms malfunction in chronic liver diseases (CLDs), which fuels disease progression. CLDs account for 2 million deaths per year worldwide. Pathophysiological studies with clinical correlation have shown evidence of deviation of normal regenerative mechanisms and its contribution to fueling fibrosis and disease progression. However, we lack realistic in vitro models that can allow experimental manipulation for mechanistic understanding of liver regeneration in CLDs and testing of candidate drugs. In this review, we aim to provide the framework for building appropriate organotypic models for dissecting regenerative responses in CLDs, with the focus on non-alcoholic steatohepatitis (NASH). By drawing parallels with development and hepatectomy, we explain the selection of critical components such as cells, signaling, and, substrate-driven biophysical cues to build an appropriate CLD model. We highlight the organoid-based organotypic models available for NASH disease modeling, including organ-on-a-chip and 3D bioprinted models. With the focus on bioprinting as a fabrication method, we prescribe building in vitro CLD models and testing schemes for exploring the regenerative responses in the bioprinted model.

Exosomes are nanosized extracellular vesicles (EVs) that participate in intercellular communication through surface proteins and the delivery of internal cargo. The exosomes have gained attention for their potential as disease biomarkers and therapeutic agents. The therapeutic ability of exosomes has been verified by copious previous studies. Effective methods for extensive clinical applications are being researched for exosome-based regenerative therapies, including the application of 3D cultures to enhance exosome production and secretion, which can resolve limited exosome secretion from the parent cells. Cell culture has emerged as a crucial approach for biomedical research because of its many benefits. Both well-established continuous cell lines and primary cell cultures continue to be invaluable for basic research and clinical application. Previous studies have shown that three-dimensional cultured exosomes (3D-Exo) improve therapeutic properties and yields compared with traditional culture systems. Since the majority of studies have focused on exosomes derived from mesenchymal stem cells (MSC-Exo), this review will also concentrate on MSC-Exo. In this review, we will summarize the advantages of 3D-Exo and introduce the 3D culture system and methods of exosome isolation, providing scientific strategies for the diagnosis, treatment, and prognosis of a wide variety of diseases.

Small-cell lung cancer (SCLC) accounts for 15% of lung cancers and has a dismal prognosis due to early dissemination and acquired chemoresistance. The initial good response to chemotherapy is followed by refractory relapses within 1-2 years. Mechanisms leading to chemoresistance are not clear and progress is poor.

This article reviews the current evidence of the resistance of SCLCs at the cellular level including alteration of key proteins and the possible presence of cancer stem cells (CSCs). Without compelling evidence for cellular mechanisms and clinical failures of novel approaches, the study of SCLC has advanced to the role of 3D tumor cell aggregates in chemoresistance.

The scarcity of viable tumor specimen from relapsed SCLC patients has hampered the investigations of acquired chemoresistance but a panel of nine SCLC circulating tumor cell (CTC) cell lines have revealed characteristics of SCLC in the advanced refractory states. The chemoresistance of relapsed SCLC seems to be linked to the spontaneous formation of large spheroids, termed tumorospheres, which contain resistant quiescent and hypoxic cells shielded by a physical barrier. So far, drugs to tackle large tumor spheroids are in preclinical and early clinical development.

Neural tissue engineering has emerged as a promising field that aims to create functional neural tissue for therapeutic applications, drug screening, and disease modelling. It is becoming evident in the literature that this goal requires development of three-dimensional (3D) constructs that can mimic the complex microenvironment of native neural tissue, including its biochemical, mechanical, physical, and electrical properties. These 3D models can be broadly classified as self-assembled models, which include spheroids, organoids, and assembloids, and engineered models, such as those based on decellularized or polymeric scaffolds. Self-assembled models offer advantages such as the ability to recapitulate neural development and disease processes in vitro, and the capacity to study the behaviour and interactions of different cell types in a more realistic environment. However, self-assembled constructs have limitations such as lack of standardised protocols, inability to control the cellular microenvironment, difficulty in controlling structural characteristics, reproducibility, scalability, and lengthy developmental timeframes. Integrating biomimetic materials and advanced manufacturing approaches to present cells with relevant biochemical, mechanical, physical, and electrical cues in a controlled tissue architecture requires alternate engineering approaches. Engineered scaffolds, and specifically 3D hydrogel-based constructs, have desirable properties, lower cost, higher reproducibility, long-term stability, and they can be rapidly tailored to mimic the native microenvironment and structure. This review explores 3D models in neural tissue engineering, with a particular focus on analysing the benefits and limitations of self-assembled organoids compared with hydrogel-based engineered 3D models. Moreover, this paper will focus on hydrogel based engineered models and probe their biomaterial components, tuneable properties, and fabrication techniques that allow them to mimic native neural tissue structures and environment. Finally, the current challenges and future research prospects of 3D neural models for both self-assembled and engineered models in neural tissue engineering will be discussed.

A three-dimensional (3D) cell culture can more precisely mimic tissues architecture and functionality, being a promising alternative model to study disease pathophysiology and drug screening. Chagas disease (CD) is a neglected parasitosis that affects 7 million people worldwide. Trypanosoma cruzi's (T. cruzi) mechanisms of invasion/persistence continue to be elucidated. Benznidazole (BZ) and Nifurtimox (NF) are trypanocidal drugs with few effects on the clinical manifestations of the chronic disease. Chronic Chagas cardiomyopathy (CCC) is the main manifestation of CD due to its frequency and severity. The development of fibrosis and hypertrophy in cardiac tissue can lead to heart failure and sudden death. Thus, there is an urgent need for novel therapeutic options. Our group has more than fifteen years of expertise using 3D primary cardiac cell cultures, being the first to reproduce fibrosis and hypertrophy induced by T. cruzi infection in vitro. These primary cardiac spheroids exhibit morphological and functional characteristics that are similar to heart tissue, making them an interesting model for studying CD cardiac fibrosis. Here, we aim to demonstrate that our primary cardiac spheroids are great preclinical models which can be used to develop new insights into CD cardiac fibrosis, presenting advances already achieved in the field, including disease modeling and drug screening.

Fibroblastic reticular cells (FRCs) are stromal cells (SCs) that can be isolated from lymph node (LN) biopsies. Studies have shown that these nonhematopoietic cells have the capacity to shape and regulate adaptive immunity and can become a form of personalized cell therapy. Successful translational efforts, however, require the cells to be formulated as injectable units, with their native architecture preserved. The intrinsic reticular organization of FRCs, however, is lost in the monolayer cultures. Organizing FRCs into three-dimensional (3D) clusters would recapitulate their structural and functional attributes. Herein, we report a scaffolding method based on the self-assembling peptide (SAP) EAKII biotinylated at the N-terminus (EAKbt). Cross-linking with avidin transformed the EAKbt fibrils into a dense network of coacervates. The combined forces of fibrillization and bioaffinity interactions in the cross-linked EAKbt likely drove the cells into a cohesive 3D reticula. This facile method of generating clustered FRCs (clFRCs) can be completed within 10 days. In vitro clFRCs attracted the infiltration of T cells and rendered an immunosuppressive milieu in the cocultures. These results demonstrate the potential of clFRCs as a method for stromal cell delivery.

Aim: Phenol red is commonly used in cell culture media, but can be detrimental to bioanalysis of in vitro samples as it may impact instrument reliability. Many researchers do their final stage of culture in 'phenol red free' media, but in collaborative work this is not always feasible.Materials & methods: A comparison was made between typical extraction methods to reduce phenol red matrix interferences, including organic solvent precipitation and solid phase extraction.Results: The final method was demonstrated to be precise and accurate for the measurement of a target analyte by LC-MS/MS, and was applied to an in vitro ADC deconjugation study.Conclusion: This method allows for for continued bioanalytical support of in vitro models used in drug development.

The tumor microenvironment (TME) of pancreatic ductal adenocarcinoma (PDAC) is characterized by deposition of desmoplastic matrix (including collagen and hyaluronic acid). And the interactions between tumor-associated macrophages (TAMs) and tumor cells play a crucial role in progression of PDAC. Hence, the appropriate model of tumor cell-macrophage interaction within the unique PDAC TME is of significantly important. To this end, a 3D tumor niche based on dual-crosslinking gelatin methacrylate and hyaluronic acid methacrylate hydrogels was constructed to simulate the desmoplastic tumor matrix with matching compressive modulus and composition. The bionic 3D tumor niche creates an immunosuppressive microenvironment characterized by the downregulation of M1 markers and upregulation of M2 markers in TAMs. Mechanistically, RNA-seq analysis revealed that the PI3K-AKT signaling pathway might modulate the phenotypic balance and recruitment of macrophages through regulating SELE and VCAM-1. Furthermore, GO and GSEA revealed the biological process of leukocyte migration and the activation of cytokine-associated signaling were involved. Finally, the 3D tumor-macrophage niches with three different ratios were fabricated which displayed increased M2-like polarization and stemness. The utilization of the 3D tumor niche has the potential to provide a more accurate investigation of the interplay between PDAC tumor cells and macrophages within an in vivo setting.

In this study, we designed the association of the organoselenium compound 5'-Seleno-(phenyl)-3'-(ferulic-amido)-thymidine (AFAT-Se), a promising innovative nucleoside analogue, with the antitumor drug paclitaxel, in poly(ε-caprolactone) (PCL)-based nanoparticles (NPs). The nanoprecipitation method was used, adding the lysine-based surfactant, 77KS, as a pH-responsive adjuvant. The physicochemical properties presented by the proposed NPs were consistent with expectations. The co-nanoencapsulation of the bioactive compounds maintained the antioxidant activity of the association and evidenced greater antiproliferative activity in the resistant/MDR tumor cell line NCI/ADR-RES, both in the monolayer/two-dimensional (2D) and in the spheroid/three-dimensional (3D) assays. Hemocompatibility studies indicated the safety of the nanoformulation, corroborating the ability to spare non-tumor 3T3 cells and human mononuclear cells of peripheral blood (PBMCs) from cytotoxic effects, indicating its selectivity for the cancerous cells. Furthermore, the synergistic antiproliferative effect was found for both the association of free compounds and the co-encapsulated formulation. These findings highlight the antitumor potential of combining these bioactives, and the proposed nanoformulation as a potentially safe and effective strategy to overcome multidrug resistance in cancer therapy.

Developing in vitro models that accurately mimic the microenvironment of biological structures or processes holds substantial promise for gaining insights into specific biological functions. In the field of tissue engineering and regenerative medicine, in vitro models able to capture the precise structural, topographical, and functional complexity of living tissues, prove to be valuable tools for comprehending disease mechanisms, assessing drug responses, and serving as alternatives or complements to animal testing. The choice of the right biomaterial and fabrication technique for the development of these in vitro models plays an important role in their functionality. In this sense, elastin-like recombinamers (ELRs) have emerged as an important tool for the fabrication of in vitro models overcoming the challenges encountered in natural and synthetic materials due to their intrinsic properties, such as phase transition behavior, tunable biological properties, viscoelasticity, and easy processability. In this review article, we will delve into the use of ELRs for molecular models of intrinsically disordered proteins (IDPs), as well as for the development of in vitro 3D models for regenerative medicine. The easy processability of the ELRs and their rational design has allowed their use for the development of spheroids and organoids, or bioinks for 3D bioprinting. Thus, incorporating ELRs into the toolkit of biomaterials used for the fabrication of in vitro models, represents a transformative step forward in improving the accuracy, efficiency, and functionality of these models, and opening up a wide range of possibilities in combination with advanced biofabrication techniques that remains to be explored.

Diabetes is a global problem that threatens human health. Cell therapy methods using stem cells, and tissue engineering of pancreatic islets as new therapeutic approaches have increased the chances of successful diabetes treatment. In this study, to differentiate Wharton's Jelly-derived mesenchymal stem cells (WJ-MSCs) into insulin-producing cells (IPCs) with improved maturity, and function, platelet-rich plasma (PRP)-Polyvinylpyrrolidone (PVP)-Polycaprolactone (PCL)/PCL scaffold was designed. The two-dimensional (2D) control group included cell culture without differentiation medium, and the experimental groups included 2D, and three-dimensional (3D) groups with pancreatic beta cell differentiation medium. WJ-MSCs-derived IPCs on PRP-PVP-PCL/PCL scaffold took round cluster morphology, the typical pancreatic islets morphology. Real-time PCR, immunocytochemistry, and flowcytometry data showed a significant increase in pancreatic marker genes in WJ-MSCs-derived IPCs on the PRP-PVP-PCL/PCL scaffold compared to the 2D-experimental group. Also, using the ELISA assay, a significant increase in the secretion of insulin, and C-peptide was measured in the WJ-MSCs-derived IPCs of the 3D-experimental group compared to the 2D experimental group, the highest amount of insulin (38 µlU/ml), and C-peptide (43 pmol/l) secretion was in the 3D experimental group, and in response to 25 mM glucose solution, which indicated a significant improvement in the functional level of the WJ-MSCs-derived IPCs in the 3D group. The results showed that the PRP-PVP-PCL/PCL scaffold can provide an appropriate microenvironment for the engineering of pancreatic islets, and the generation of IPCs.

Axon regeneration and Schwann cell proliferation are critical processes in the repair and functional recovery of damaged neural tissues. Biomaterials can play a crucial role in facilitating cell proliferative processes that can significantly impact the target tissue repair. Chemical decellularization and supercritical fluid-based decellularization methods are similar approaches that eliminate DNA from native tissues for tissue-mimetic biomaterial production by using different solvents and procedures to achieve the final products. In this study, we conducted a comparative analysis of these two methods in the context of nerve regeneration and neuron cell differentiation efficiency. We evaluated the efficacy of each method in terms of biomaterial quality, preservation of extracellular matrix components, promotion of neuronal cell differentiation and nerve tissue repair ability in vivo. Our results indicate that while both methods produce high-quality biomaterials, supercritical fluid-based methods have several advantages over conventional chemical decellularization, including better preservation of extracellular matrix components and mechanical properties and superior promotion of cellular responses. We conclude that supercritical fluid-based methods show great promise for biomaterial production for nerve regeneration and neuron cell differentiation applications.

Owing to their advantages such as great specificity, sensitivity, rapidity, and possibility of noninvasive and real-time monitoring, electrochemical cell-based biosensors (ECBBs) have been a powerful tool for food analysis encompassing the areas of nutrition, flavor, and safety. Notably, the distinctive biological relevance of ECBBs enables them to mimic physiological environments and reflect cellular behaviors, leading to valuable insights into the biological function of target components in food. Compared with previous reviews, this review fills the current gap in the narrative of ECBB construction strategies. The review commences by providing an overview of the materials and configuration of ECBBs, including cell types, cell immobilization strategies, electrode modification materials, and electrochemical sensing types. Subsequently, a detailed discussion is presented on the fabrication strategies of ECBBs in food analysis applications, which are categorized based on distinct signal sources. Lastly, we summarize the merits, drawbacks, and application scope of these diverse strategies, and discuss the current challenges and future perspectives of ECBBs. Consequently, this review provides guidance for the design of ECBBs with specific functions and promotes the application of ECBBs in food analysis.

In vitro cell culture is a well-established technique present in numerous laboratories in diverse areas. In reproduction, gametes, embryos, and reproductive tissues, such as the ovary and endometrium, can be cultured. These cultures are essential for embryo development studies, understanding signaling pathways, developing drugs for reproductive diseases, and in vitro embryo production (IVP). Although many culture systems are successful, they still have limitations to overcome. Three-dimensional (3D) culture systems can be close to physiological conditions, allowing greater interaction between cells and cells with the surrounding environment, maintenance of the cells' natural morphology, and expression of genes and proteins such as in vivo. Additionally, three-dimensional culture systems can stimulated extracellular matrix generating responses due to the mechanical force produced. Different techniques can be used to perform 3D culture systems, such as hydrogel matrix, hanging drop, low attachment surface, scaffold, levitation, liquid marble, and 3D printing. These systems demonstrate satisfactory results in follicle culture, allowing the culture from the pre-antral to antral phase, maintaining the follicular morphology, and increasing the development rates of embryos. Here, we review some of the different techniques of 3D culture systems and their applications to the culture of follicles and embryos, bringing new possibilities to the future of assisted reproduction.

In plant tissue culture, callus formation serves as a crucial mechanism for regenerating entire plants, enabling the differentiation of diverse tissues. Researchers have extensively studied the influence of media composition, particularly plant growth regulators, on callus behavior. However, the impact of the physical properties of the media, a well-established factor in mammalian cell studies, has received limited attention in the context of plant tissue culture. Previous research has highlighted the significance of gelling agents in affecting callus growth and differentiation, with Agar, Phytagel, and Gelrite being the most used options. Despite their widespread use, a comprehensive comparison of their physical properties and their subsequent effects on callus behavior remains lacking. Our study provides insights into optimizing plant tissue culture media by analyzing the physical properties of gelling agents and their impact on callus induction and differentiation. We compared the phenotypes of calli grown on media composed of these different gelling agents and correlated them to the physical properties of these media. We tested water retention, examined pore size using cryo-SEM, measured the media mechanical properties, and studied diffusion characteristics. We found that the mechanical properties of the media are the only quality correlated with callus phenotype.

Organoids significantly advanced our comprehension of organ development, function, and disease modeling. This Perspective underscores the potential of heart-kidney-connected organoids in understanding the intricate relationship between these vital organs, notably the cardiorenal syndrome, where dysfunction in one organ can negatively impact the other. Conventional models fall short in replicating this complexity, necessitating an integrated approach. By co-culturing heart and kidney organoids, combined with microfluidic and 3D bioprinting technologies, a more accurate representation of in vivo conditions can be achieved. Such interconnected systems could revolutionize our grasp of multi-organ diseases, drive drug discovery by evaluating therapeutic agents on both organs simultaneously, and reduce the need for animal models. In essence, heart-kidney-connected organoids present a promising avenue to delve deeper into the pathophysiology underlying cardiorenal disorders, bridging existing knowledge gaps, and advancing biomedical research.

The three-dimensional (3D) cell culture system is being employed more frequently to investigate cell engineering and tissue repair due to its close mimicry of in vivo microenvironments. In this study, we developed natural biomaterials, including hyaluronic acid, alginate, and gelatin, to mimic the creation of a 3D human mesenchymal stem cell (hMSC) extracellular environment and selected hydrogels with high proliferation capacity for 3D MSC culture. Human mesenchymal stem cells were encapsulated within hydrogels, and an investigation was conducted into the effects on cell viability and proliferation, stemness properties, and telomere activity compared to the 2D monolayer culture. Hydrogel characterization, cell proliferation, Live/Dead cell viability assay, gene expression, telomere relative length, and MSC stemness-related proteins by immunofluorescence staining were examined. The results showed that 3D alginate-hyaluronic acid (AL-HA) hydrogels increased cell proliferation, and the cells were grown as cellular spheroids within hydrogels and presented a high survival rate of 77.36% during the culture period of 14 days. Furthermore, the 3D alginate-hyaluronic acid (AL-HA) hydrogels increased the expression of stemness-related genes (OCT-4, NANOG, SOX2, and SIRT1), tissue growth and development genes (YAP and TAZ), and cell proliferation gene (Ki67) after culture for 14 days. Moreover, the telomere activity of the 3D MSCs was enhanced, as indicated by the upregulation of the human telomerase reverse transcriptase gene (hTERT) and the relative telomere length (T/S ratio) compared to the 2D monolayer culture. Altogether, these data suggest that the 3D alginate-hyaluronic acid (AL-HA) hydrogels could serve as a promising material for maintaining stem cell properties and might be a suitable carrier for tissue engineering proposals.

Shape-controlled 3D tissues resemble natural living tissues in human and animal bodies and are essential materials for developing and improving technologies in regenerative medicine, drug discovery, and biological robotics. In previous studies, shape-controlled 3D tissues were fabricated using scaffold structures or 3D bioprinting techniques. However, controlling the shape of 3D tissues without leaving non-natural materials inside the 3D tissue and efficiently fabricating them remains challenging. In this paper, we propose a novel method for fabricating shape-controlled 3D tissues free of non-natural materials using a flexible high-porosity porous structure (HPPS). The HPPS consisted of a micromesh with pore sizes of 14.87 ± 1.83 μm, lattice widths of 2.24 ± 0.10 μm, thicknesses of 9.96 ± 0.92 μm, porosity of 69.06 ± 3.30%, and an I-shaped microchamber of depth 555.26 ± 11.17 μm. U-87 human glioma cells were cultured in an I-shaped HPPS microchamber for 48 h. After cultivation, the 3D tissue was released within a few seconds while maintaining its I-shape. Specific chemicals, such as proteolytic enzymes, were not used. Moreover, the viability of the released cells composed of shape-controlled 3D tissues free of non-natural materials was above 90%. Therefore, the proposed fabrication method is recommended for shape-controlled 3D tissues free of non-natural materials without applying significant stresses to the cells.

Three-dimensional (3D) cell cultures have garnered significant attention in biomedical research due to their ability to mimic the in vivo cellular environment more accurately. The formation of 3D cell spheroids using hanging drops has emerged as a cost-effective and crucial method for generating uniformly-sized spheroids. This study aimed to validate the potential of a tip-refill wafer (TrW), a disposable laboratory item used to hold pipette tips, in facilitating 3D cell culture. The TrW allows for easy generation of hanging drops by pipetting the solution into the holes of the wafer. The mechanical stability of the hanging drops is ensured by the surface wettability and thickness of the TrW. Hanging drops containing 60-µL of solution remained securely attached to the TrW even when subjected to orbital shaking at 210 rpm. The exceptional resistance to mechanical shaking enabled the use of inertial focusing to facilitate spheroid formation. This was demonstrated through live/dead cell staining, quantitative polymerase chain reaction (qPCR) analysis, and cytoskeleton staining, which revealed that horizontal orbiting at 60 rpm for 15 min promoted cell aggregation and ultimately led to the formation of 3D spheroids. The spheroid harvest rate is 96.1% ± 3.5% across three TrWs, each containing 60 hanging drops. In addition to generating mono-culture 3D spheroids, the TrW-based hanging drop platform also enables the formation of multicellular spheroids, and on-demand pairing and fusion of spheroids. The TrW is a disposable item that does not require any fabrication or surface modification procedures, further enhancing its application potential in conventional biological laboratories.