Cells exhibit remarkable sensitivity to the mechanical properties of their surrounding matrix, particularly stiffness changes, a phenomenon known as cellular mechanotransduction. In vivo, tissues exhibit a wide range of stiffness, from kilopascals (kPa) to megapascals (MPa), which can alter with aging and disease. Traditional cell culture methods employ plastic substrates with stiffness in the gigapascal range, which does not accurately mimic the physiological conditions of most biological tissues. Therefore, employing substrates that can be engineered to span a wide range of stiffnesses, closely resembling the native tissue environment, is crucial for obtaining results that more accurately reflect cellular responses in vivo. Polydimethylsiloxane (PDMS) substrates are widely used in cell culture for their ability to simulate tissue stiffness, but their optimization presents several challenges. Fabrication requires precise control over mixing, weighing, and curing to ensure reproducible mechanical properties. Inconsistent preparation can lead to improperly cured PDMS substrates, compromising experimental outcomes. Additionally, PDMS's inherent hydrophobicity poses challenges for cell attachment, necessitating surface modifications to enhance adhesion. Moreover, the risk of contamination during the sterilization process necessitates stringent protocols to maintain cell culture integrity. These challenges are further compounded by substrate autofluorescence which can cause difficulties when imaging cells. The aim of this study is to develop a standardized method for fabricating PDMS substrates with tuneable stiffness, ranging from kPa to MPa, suitable for diverse cell types using standard laboratory equipment. This method aims to minimize the complexity and equipment required for PDMS fabrication, ensuring reproducibility and ease of use. Achieving consistent and contaminant-free PDMS substrates will facilitate a broader adoption of these substrates in mechanobiology research and improve the relevance of in vitro models to in vivo conditions. Ultimately, contributing to a more comprehensive understanding of cellular responses to mechanical cues in health and disease.

Hydrogels are one of the most attractive biomaterials, used in both three-dimensional (3D) and in vivo cultures. They facilitate the reconstruction of tissue microenvironments by preserving the spatial arrangement of cells, cell-cell interactions, and functional dynamics in the tissue. In this work, the long-term effect of alginate hydrogel on cell culture and the possibility of rapid cell recovery by dissolving the hydrogel were investigated. Mouse glial-restricted progenitors (GRPs) and porcine mesenchymal stem cells (MSCs) were suspended in hydrogels; their metabolic activity, viability, and expression of genes, which are involved in oxidative stress, apoptosis, proliferation, migration, and differentiation, were assessed using quantitative polymerase chain reaction (qPCR). The concentration that was able to dissolve the hydrogel and was the least harmful to the cells was 0.005 M ethylenediaminetetraacetic acid (EDTA). The metabolism of both cell types was reduced from the beginning of the experiment to day 3. From day 7 to the end of the experiment, the normalization of the GRP metabolism was observed, in contrast to the MSCs. For the apoptosis-related genes, caspase 3, 7, and B-cell leukemia (Casp3, Casp 7, Bcl2) were increased in GRPs and MSCs on days 0 and 1. After 3 and 7 days, an increase in the expression of oxidative stress genes (nuclear factor of activated T-cells 5-NFAT5 and autophagy-related 14-ATG14) was observed in cells cultured in calcium chloride (CaCl2). GRPs cultured in calcium alginate (CaM) were not affected and, remarkably, showed increased Antigen Kiel 67 (Ki67) levels after 30 days. In conclusion, alginate hydrogels provide an excellent environment for stem cell culture in 3D for a longer period of time, but this is dependent on the cell type. Therefore, an individual approach to cell culture is necessary, taking into account the requirements of the cells to be used.

Bioprinting of microtissues has become a standard technique in medical and biotechnological research, offering a more accurate replication of thein vivosetting than conventional 2D cell culture. However, widespread adoption is limited by the absence of a universally accepted printing benchmark-common in standard fused deposition modeling (FDM) printing, as well as the high cost and restricted customizability of commercial bioprinters. This study introduces a method to convert a standard FDM printer into a bioprinter. All cell-contacting components are biocompatible and autoclavable, while the printer body can be UV-sanitized. Using a heated FDM printhead, we used the thermal properties of alginate-gelatin bioinks to achieve high-resolution 3D printing. A key achievement was the developed print quality index (PQI) method, which correlates nozzle temperature with bioink flow behavior, streamlining optimization of slicer settings. Guided by PQI, we reproducibly bioprinted complex alginate-gelatin structures with high quality and dimensional/geometric accuracy. A case study using recombinant HuH7EGFPcell-laden hydrogels demonstrated long-term cell proliferation, confirming high viability. Given its efficiency, the PQI method has the potential to become the missing printing benchmark for slicer optimization in bioprinting. The presented approach significantly advances the accessibility of sophisticated bioprinting technology to interested research groups worldwide.

Nanofiber-embedded 3D hydrogel constructs have garnered significant attention due to their versatile applications in drug delivery, cell therapy, tissue engineering, and regenerative medicine. These constructs are especially prized for their capacity to mimic the composition of the extracellular matrix (ECM) found in living tissues and organs. The unique chemical and mechanical properties of hydrogel microcapsules have made them particularly notable among various biomaterial constructs for their effectiveness in cell encapsulation, which aims to improve cell growth and proliferation. In this study, we developed alginate hydrogel microcapsules embedded with chitin nanofibrils, using divalent calcium ions and trivalent iron ions as crosslinking agents. An electrostatic encapsulation technique was utilized to create microcapsules with diameters ranging from 200-500 μm, and their physicochemical properties, rheological properties, size, and mechanical stability were evaluated. The rheological analysis demonstrated that the Fe3+ crosslinked hydrogel (AF0) and Fe3+/Ca2+ cross-linked hydrogel (AFC) have higher storage modulus than the Ca2+ crosslinked hydrogel (AC0). Additionally, FTIR analyses of AF0 and AFC demonstrated a broader -O-H stretching peak compared to that of AC0, suggesting that more hydroxyl groups of alginate chains are involved in crosslinking with ferric ions exhibiting extended mechanical stability compared to those crosslinked with calcium ions under in vitro physiological conditions. We also investigated the cellular responses to the composite hydrogels crosslinked with these divalent and trivalent metal ions through in vitro studies involving the seeding and encapsulation of NIH/3T3 fibroblast cells. Remarkably, both types of crosslinked microcapsules maintained excellent cell viability for up to 5 days. Our in vitro scratch assay demonstrated that media extracted from AF0 microcapsules facilitated faster wound closure compared to that extracted from AC0, suggesting that hydrogels crosslinked with Fe3+ ions promote enhanced cellular proliferation. These results suggest that calcium and ferric ion crosslinked alginate-chitin composite microcapsules provide a promising platform for developing 3D hydrogel constructs suitable for various biomedical applications, including wound healing models, tissue engineering, and drug toxicity testing.

Biofabrication and three-dimensional (3D) bioprinting enable precise spatial arrangement of cells within biomaterial scaffolds. We developed an alginate-based and Förster resonance energy transfer (FRET)-responsive "turn-on" reporter ink platform to enable real-time monitoring of matrix metalloproteinase (MMP) activity. Three distinct MMP-cleavable turn-on peptide reporters were synthesized and characterized for their cell-specific cleavage profiles using recombinant MMPs, cell-derived media, and different cell cultures (NIH3T3, HEK293, and MelHo). All turn-on reporters were covalently and site-specifically incorporated into alginate dialdehyde (ADA) to yield an MMP reporter ink. The ADA reporter ink with an MMP 13 turn-on reporter was responsive to all tested cell types over time within the cast bulk constructs. The ADA reporter ink material blended with gelatin had comparable print resolution and structural fidelity as observed for ADA. The extrusion-based bioprinted MelHo cell grids, measuring 2 × 2 cm2 and containing 1 × 106 cells/mL, exhibited MMP activity responses comparable to those of the casted reporter ink system, with a 3-fold increase observed at 24 h. This study introduces a versatile, FRET-based alginate bioink platform for the real-time monitoring of MMP activities, expanding the toolkit to understand cellular performance in bioprinted 3D constructs.

Angiogenesis-osteogenesis coupling is a crucial process in bone tissue engineering, requiring a suitable material structure for vessel growth. Recently, the 3D culture system has gained significant attention due to its benefits in cell growth, proliferation and tissue regeneration. Its most notable advantage is its ECM-like function, which supports endothelial cell adhesion and facilitates the formation of vascular-like networks-crucial for angiogenesis-osteogenesis coupling. Hydrogels, with their highly hydrophilic polymer network resembling the extracellular matrix, make the 3D gel culture system an ideal approach for angiogenesis due to its cellular integrity and adjustable properties. This article reviews the current use of 3D gel culture systems in bone tissue engineering, covering substrates, characteristics and processing technologies, thereby offering readers profound insights into these systems.

Biomimetic hydrogels enable biochemical, cell biology, and tissue-like studies in the third dimension. Smart hydrogels are also frequently used in tissue engineering and as drug carriers for intra- or extracutaneous regenerative medicine. They have also been studied in bio-sensor development, 3D cell culture, and organoid growth optimization. Yet, many hydrogel types, adjuvant components, and cross-linking methods have emerged over decades, diversifying and complexifying such studies. Here, an evaluative overview is provided, mapping potential applications to the corresponding hydrogel tuning. Strikingly, hydrogels are ideal for studying locoregional therapy modalities, such as cold medical gas plasma technology. These partially ionized gases produce various reactive oxygen species (ROS) types along with other physico-chemical components such as ions and electric fields, and the spatio-temporal effects of these components delivered to diseased tissues remain largely elusive to date. Hence, this work outlines the promising applications of hydrogels in biomedical research in general and cold plasma science in particular and underlines the great potential of these smart scaffolds for current and future research and therapy.

This study investigates the synthesis of potato starch elastomers reinforced with silicon dioxide (SiO2) and citric acid as a crosslinking agent to enhance their mechanical and barrier properties. Surface morphology analysis using optical microscopy revealed that pure potato starch films had uneven surfaces. However, higher SiO2 concentrations increased roughness, while citric acid crosslinked films displayed smoother surfaces overall. Water vapor transmission rates (WVTR) indicated that native starch films were highly hydrophilic, while SiO2 incorporation and citric acid crosslinking significantly reduced WVTR of 17% (30% lower than native film), enhancing the barrier properties. Tensile strength testing revealed that citric acid crosslinking increased the tensile strength by 25%, while SiO2 further reinforced the films but decreased elasticity by 15%. SiO2 had little impact on degradation rates, while citric acid crosslinking delayed microbial growth, extending film longevity by 20%. Biocompatibility assays using SiHa, HT-29, and HEK 293 cell lines revealed that the films had varying degrees of cell confluency. Films with both SiO2 and citric acid showed improved confluency (20% higher) compared to films containing only SiO2. However, citric acid alone resulted in the highest confluency (95% viability), suggesting its significant role in biocompatibility. This eco-friendly approach demonstrates substantial advancements in film properties, offering potential applications in diverse biomedical industries.

Cultured meat aims to produce meat mass by culturing cells and tissues based on the muscle regeneration mechanism, and is considered an alternative to raising and slaughtering livestock. Hydrogel building blocks are commonly used as substrates for cell culture in tissue engineering and cultured meat because of their high water content, biocompatibility, and similar three-dimensional (3D) environment to the cellular niche in vivo. With the characteristics of precise manipulation of fluids, microfluidics exhibits advantages in the fabrication of building blocks with different structures and components, which have been widely applied in tissue regeneration. Microfluidic building blocks show promising prospects in the field of cultured meat; however, few reviews on the application of microfluidic building blocks in cultured meat have been published. This review outlines the recent status and prospects of the use of microfluidic building blocks in cultured meat. Starting with the introduction of cells and materials for cultured meat tissue construction, we then describe the diverse structures of the fabricated building blocks, including microspheres, microfibers, and microsphere-microfiber hybrid systems. Next, the stacking strategies for tissue construction are highlighted in detail. Finally, challenges and future prospects for developing microfluidic building blocks for cultured meat are discussed.

In recent years, the realm of tissue regeneration experienced significant advancements, leading to the development of innovative therapeutic agents. The systemic delivery of mesenchymal stem cells (MSCs) emerged as a promising strategy for promoting tissue regeneration. However, this approach is hindered by hurdles such as poor cell survival, limited cell propagation, and inadequate cell integration. Decellularized extracellular matrix (dECM) hydrogel serves as an innovative carrier that protects MSCs from the detrimental effects of the hostile microenvironment, facilitates their localization and retention at the injection site, and preserves their viability. Regarding its low immunogenicity, low cytotoxicity, high biocompatibility, and its ability to mimic natural extracellular matrix (ECM), this natural hydrogel offers a new avenue for systemic delivery of MSCs. This review digs into the properties of dECM hydrogels (dECMHs), the methods employed for decellularization and the utilization of dECMH as carriers for various types of MSCs for tissue regeneration purposes. This review also sheds light on the benefits of hybrid hydrogels composed of dECMH and other components such as proteins and polysaccharides. By addressing the limitations of conventional hydrogels and enhancing efficacy of cell therapy, dECMH opens new pathways for the future of tissue regeneration.

Developing effective drug screening methods for type 2 diabetes requires physiologically relevant models. Traditional 2D cell cultures have limitations in replicating in vivo conditions, leading to challenges in assessing drug efficacy. To overcome these issues, we developed a 3D artificial muscle model that induces insulin resistance, a hallmark of type 2 diabetes. Using C2C12 myoblasts cultured in a scaffold of 1% alginate and 1 mg/mL collagen type 1, we optimized conditions for differentiation and structural stability. Insulin resistance was induced using palmitic acid (PA), and glucose uptake was assessed using the fluorescent glucose analog 2-NBDG. The 3D model demonstrated superior glucose uptake responses compared with 2D cultures, with a threefold increase in insulin-stimulated glucose uptake on days 4 and 8 of differentiation. Induced insulin resistance was observed with 0.1 mM PA, which maintained cell viability and differentiation capacity. The model was validated through comparative drug screening using rosiglitazone and metformin, as well as 165 candidate compounds provided by Korea Chemical Bank. Drug screening revealed that three out of five hit compounds identified in both 2D and 3D models exhibited greater efficacy in 3D cultures, with results consistent with ex vivo assays using mouse soleus muscle. This model closely mimics in vivo conditions, offering a robust platform for type 2 diabetes drug discovery while supporting ethical research practices.

Invariant Natural Killer T (iNKT) cells are a unique subset of T cells that bridge innate and adaptive immunity, displaying potent anti-tumor properties through cytokine secretion, direct cytotoxicity, and recruitment of immune effector cells such as CD8+ T cells and NK cells. Despite their therapeutic potential, the immunosuppressive tumor microenvironment (TME), characterized by regulatory T cells, myeloid-derived suppressor cells (MDSCs), and tumor-associated macrophages (TAMs), limits iNKT cell efficacy. Patient-derived organoid (PDO) platforms provide an innovative model for dissecting these complex interactions and evaluating strategies to reinvigorate iNKT cell functionality within the TME. PDOs closely mimic the genetic, phenotypic, and structural characteristics of primary tumors, enabling the study of tumor-immune dynamics. Integrating iNKT cells into PDOs offers a robust platform for investigating CD1d-mediated interactions, Th1-biased immune responses driven by glycolipid analogs like α-GalCer, and combination therapies such as immune checkpoint inhibitors. Additionally, PDO systems can assess the effects of metabolic modulation, including reducing lactic acid accumulation or targeting glutamine pathways, on enhancing iNKT cell activity. Emerging innovations, such as organoid-on-a-chip systems, CRISPR-Cas9 gene editing, and multi-omics approaches, further expand the potential of PDO-iNKT platforms for personalized immunotherapy research. Although the application of iNKT cells in PDOs is still undeveloped, these systems hold immense promise for bridging preclinical studies and clinical translation. By addressing the challenges of the TME and optimizing therapeutic strategies, PDO-iNKT platforms offer a transformative avenue for advancing cancer immunotherapy and personalized medicine.

In this preliminary study, the long-term effects of calcium chloride crosslinking concentration on viability of 16HBE14o- human bronchial epithelial cells embedded in alginate-extracellular matrix (ECM) or alginate-methylcellulose-ECM hydrogels have been investigated. There is currently a limited understanding regarding the effects of crosslinking solution concentration on lung epithelial cells embedded in hydrogel. Furthermore, the effects of calcium chloride concentration in crosslinking solutions on other cell types have not been reported regarding whether the addition of viscosity and stiffness tuning agents such as methylcellulose will alter the responses of cells to changes in calcium chloride concentration in crosslinking solutions. While there were no significant effects of calcium chloride concentration on cell viability in alginate-ECM hydrogels, there is a decrease in cell viability in alginate-methylcellulose-ECM hydrogels crosslinked with 300 mM calcium chloride crosslinking solution. These findings have implications in the maintenance of 16HBE14o- 3D cultures with respect to the gelation of alginate with high concentrations of ionic crosslinking solution.

Autologous fat is widely used in soft tissue reconstruction; however, significant volume reduction owing to necrosis and degradation of the transplanted adipose tissue (AT) remains a major challenge. To address this issue, a novel live AT micro-fragment-based bio-ink (ATmf bio-ink) compatible with precision 3D printing, is developed. Live AT micro-fragments of ≈280 µm in size are prepared using a custom tissue micronizer and they are incorporated into a fibrinogen/gelatin mixture to create the ATmf bio-ink. AT micro-fragments exhibit high viability and preserve the heterogeneous cell population and extracellular matrix of the native AT. The developed bio-ink enables precise micropatterning and provides an excellent adipo-inductive microenvironment. AT grafts produced by co-printing the bio-ink with polycaprolactone demonstrate a 500% improvement in volume retention and a 300% increase in blood vessel infiltration in vivo compared with conventional microfat grafts. In vivo engraftment of AT grafts is further enhanced by using a stem cell-laden ATmf bio-ink. Last, it is successfully demonstrated that the bio-ink is enabled for the creation of clinically relevant and patient-specific AT grafts for patients undergoing partial mastectomy. This novel ATmf bio-ink for volumetric soft tissue reconstruction offers a pioneering solution for addressing the limitations of existing clinical techniques.

Three-dimensional (3D) bioprinting is considered one of the most advanced tools to build up materials for tissue engineering. The aim of this work was the design, development and characterization of a bioink composed of human mesenchymal stromal cells (hMSC) for extrusion through nozzles to create these 3D structures that might potentially be apply to replace the function of damaged natural tissue. In this study, we focused on the advantages and the wide potential of biocompatible biomaterials, such as hyaluronic acid and alginate for the inclusion of hMSC. The bioink was characterized for its physical (pH, osmolality, degradation, swelling, porosity, surface electrical properties, conductivity, and surface structure), mechanical (rheology and printability) and biological (viability and proliferation) properties. The developed bioink showed high porosity and high swelling capacity, while the degradation rate was dependent on the temperature. The bioink also showed negative electrical surface and appropriate rheological properties required for bioprinting. Moreover, stress-stability studies did not show any sign of physical instability. The developed bioink provided an excellent environment for the promotion of the viability and growth of hMSC cells. Our work reports the first-time study of the effect of storage temperature on the cell viability of bioinks, besides showing that our bioink promoted a high cell viability after being extruded by the bioprinter. These results support the suggestion that the developed hMSC-composed bioink fulfills all the requirements for tissue engineering and can be proposed as a biological tool with potential applications in regenerative medicine and tissue engineering.

Polysaccharides such as sodium alginate, pectin and gellan gum are widely used biomaterials, for their ability to easily form hydrogels in the presence of divalent metal ions, such as calcium - a process often cited as a mild crosslinking mechanism. However, when using these materials as substrates for tissue engineering, there is a lack of extensive studies that investigate the impact of elevated calcium concentrations on cell health and behaviour. In this study, we performed an in-depth exploration to understand the potential effects of raising extracellular CaCl2 on cell viability, proliferation, morphology and migration. We used an established glioblastoma (GBM) cell line (U251), human dermal fibroblasts (HDF), and murine osteoblasts (MC3T3) to assess the consequences of using CaCl2 in tissue engineered models to help reevaluate biomaterial suitability and enhance standardisation practices in the field of tissue engineering. Our findings revealed that the addition of CaCl2 induced notable morphological changes in GBM cells when cultured in 3D hydrogels with excess CaCl2 added, leading to a transition from mesenchymal to amoeboid phenotypes, even at a concentration as low as 8 mM. Furthermore, cell viability was reduced in a concentration-dependent manner across all cell types, and migration was also affected. Despite the widespread use of high CaCl2 concentrations to facilitate scaffold gelation, our research unveils that there can be significant risks to cell viability, proliferation, morphology, and migration when such practices are not preceded by cell line-specific experimentation and thorough standardization procedures. This highlights the importance of careful consideration and optimisation of CaCl2 concentration when used as a crosslinking agent for hydrogels intended for use in tissue engineering applications that demand accurate recapitulation of cellular responses and physiological conditions.

The field of oncology has been changed by the application of hydrogels. These 3D polymeric networks have demonstrated significant promise in the treatment of cancer and can boost the efficacy of conventional therapeutics including chemotherapy and immunotherapy. Noteworthy, the development of biocompatible and effective hydrogels has been of interest. In this case, alginate as a biopolymer and carbohydrate polymer has been used to modify or synthesis multifunctional nanoparticles for the treatment of human diseases, especially cancer. Therefore, highlighting the function of alginate in the development of hydrogels in cancer therapy can provide new insights for improving outcome and survival rate of patients. Alginate hydrogels improve the specific and selective delivery of cargo and therefore, they reduce the systemic toxicity of drugs, while they enhance anti-cancer activity. Alginate hydrogels protect the genes against degradation by enzymes and increase blood circulation time. The alginate hydrogels can respond to the specific stimuli in the tumor microenvironment including pH, redox and light to improve the site-specific release of cargo. The nanoparticles can be incorporated in the structure of alginate hydrogels to augment their anti-cancer activity. In addition, alginate hydrogels can accelerate immunotherapy and phototherapy through delivery of immunomodulators and photosensitizers, respectively.

In this tutorial mini-review, we explore the application of Design of Experiments (DOE) as a powerful statistical tool in biotechnology. Specifically, we review the optimization of hydrogel materials for diverse microbial applications related to green microbiology, the use of microbes to promote sustainability. Hydrogels, three-dimensional polymers networks with high water retention capabilities, are pivotal in the immobilization of microorganisms and provide a customizable environment essential for directing microbial fate. We focus on the application of DOE to precisely tailor hydrogel compositions for a range of fungi and bacteria either used for the sustainable production of chemical compounds, or the elimination of hazardous substances. We examine a variety of DOE design strategies such as central composite designs, Box-Behnken designs, and optimal designs, and discuss their strategic implementation across diverse hydrogel formulations. Our analysis explores the integral role of DOE in refining hydrogels derived from a spectrum of polymers, including natural and synthetic polymers. We illustrate how DOE facilitates nuanced control over hydrogel properties that cannot be achieved using a standard one factor at a time approach. Furthermore, this review reveals a conserved finding across different materials and applications: there are significant interactions between hydrogel parameters and cell behavior. This highlights the intricacies of cell-hydrogel interactions and the impact on hydrogel material properties and cellular functions. Lastly, this review not only highlights DOE's efficacy in streamlining the optimization of cell-hydrogel processes but also positions it as a critical tool in advancing our understanding of cell-hydrogel dynamics, potentially leading to innovative advancements in biotechnological applications and bioengineering solutions.

Plant-derived polyphenols are naturally occurring compounds widely distributed in plants. They have received greater attention in the food and pharmaceutical industries due to their potential health benefits, reducing the risk of some chronic diseases due to their antioxidant, anti-inflammatory, anticancer, cardioprotective, and neuro-action properties. Polyphenolic compounds orally administered can be used as adjuvants in several treatments but with restricted uses due to chemical instability. The review discusses the different structural compositions of polyphenols and their influence on chemical stability. Despite the potential and wide applications, there is a need to improve the delivery of polyphenolics to target the human intestine without massive chemical modifications. Oral administration of polyphenols is unfeasible due to instability, low bioaccessibility, and limited bioavailability. Nano-delivery systems based on polysaccharides (starch, pectin, chitosan, and cellulose) have been identified as a viable option for oral ingestion, potentiate biological effects, and direct-controlled delivery in specific tissues. The time and dose can be individualized for specific diseases, such as intestinal cancer. This review will address the mechanisms by which polysaccharides-based nanostructured systems can protect against degradation and enhance intestinal permeation, oral bioavailability, and the potential application of polysaccharides as nanocarriers for the controlled and targeted delivery of polyphenolic compounds.

Three-dimensional bioprinting leverages computer-aided design to construct tissues and organs with specialized bioinks. A notable biomaterial for this purpose is chitosan, a natural polysaccharide sourced from crustacean exoskeletons. Chitosan's biocompatibility, biodegradability, non-toxicity, and ability to promote cell adhesion and proliferation make it an excellent component for bioinks. Initially, the rheological properties of chitosan presented challenges for its use in bioprinting. Enhancements in its printability and stability were achieved by integrating it with other natural or synthetic polymers, facilitating its successful application in bioprinting. Chitosan-based bioinks are particularly promising for controlled drug delivery. Incorporating pharmaceuticals directly into the bioink enables the printed structures to serve as localized, sustained-release systems. This approach offers multiple advantages, including precise drug delivery to targeted disease sites, increased therapeutic efficiency, and reduced systemic side effects. Moreover, bioprinting allows for the customization of drug delivery mechanisms to meet individual patient requirements. Although there have been considerable advancements, the use of chitosan-based bioinks in drug delivery is still an emerging field. This review highlights chitosan's essential role in both systemic and localized drug delivery, underscoring its significance and discussing ongoing trends in its application for pharmaceutical purposes.

Cartilage repair necessitates regenerative medicine because of the unreliable healing mechanism of cartilage. To yield a sufficient number of cells for transplantation, chondrocytes must be expanded in culture. However, in 2D culture, chondrocytes tend to lose their distinctive phenotypes and functionalities after serial passage, thereby limiting their efficacy for tissue engineering purposes. The mechanism of dedifferentiation in 2D culture can be attributed to various factors, including abnormal nuclear strength, stress-induced mitochondrial impairment, chromatin remodeling, ERK-1/2 and the p38/mitogen-activated protein kinase (MAPK) signaling pathway. These mechanisms collectively contribute to the loss of chondrocyte phenotype and reduced production of cartilage-specific extracellular matrix (ECM) components. Chondrocyte 3D culture methods have emerged as promising solutions to prevent dedifferentiation. Techniques, such as scaffold-based culture and scaffold-free approaches, provide chondrocytes with a more physiologically relevant environment, promoting their differentiation and matrix synthesis. These methods have been used in cartilage tissue engineering to create engineered cartilage constructs for transplantation and joint repair. However, chondrocyte 3D culture still has limitations, such as low viability and proliferation rate, and also difficulties in passage under 3D condition. These indicate challenges of obtaining a sufficient number of chondrocytes for large-scale tissue production. To address these issues, ongoing studies of many research groups have been focusing on refining culture conditions, optimizing scaffold materials, and exploring novel cell sources such as stem cells to enhance the quality and quantity of engineered cartilage tissues. Although obstacles remain, continuous endeavors to enhance culture techniques and overcome limitations offer a promising outlook for the advancement of more efficient strategies for cartilage regeneration.

Alginates are salts of alginic acid derived mainly from sea algae of the genus brown algae. They are also synthesized by some bacteria. They belong to negatively charged polysaccharides exhibiting some rheological properties. High plasticity and the ability to modify the structure are the reasons for their application in numerous industries. Moreover, when in contact with the living tissue, they do not trigger an immune response, and for this reason they are the most often tested materials for medical applications. The paper discusses the latest applications, including 3D bioprinting, drug delivery systems, and sorptive properties. Recognizing alginates as biomaterials, it emphasizes the necessity for precise processing and modification to industrialize them for specific uses. This review aims to provide a thorough understanding of the advancements in alginate research, underscoring their potential for innovative applications.

Micro elastofluidics is an emerging research field that encompasses characteristics of conventional microfluidics and fluid-structure interactions. Micro elastofluidics is expected to enable practical applications, for instance, where direct contact between biological samples and fluid handling systems is required. Besides design optimization, choosing a proper material is critical to the practical use of micro elastofluidics upon interaction with biological interface and after its functional lifetime. Biodegradable polymers are one of the most studied materials for this purpose. Micro elastofluidic devices made of biodegradable polymers possess exceptional mechanical elasticity, excellent bio compatibility, and structural degradability into non-toxic products. This article provides an insightful and systematic review of the utilization of biodegradable polymers in digital and continuous-flow micro elastofluidics.

The management of vision-threatening retinal diseases remains challenging due to the lack of an effective drug delivery system. Encapsulated cell therapy (ECT) offers a promising approach for the continuous delivery of therapeutic agents without the need for immunosuppressants. In this context, an injectable and terminable collagen-alginate composite (CAC) ECT gel, designed with a Tet-on pro-caspase-8 system, was developed as a safe intraocular drug delivery platform for the sustained release of glial-cell-line-derived neurotrophic factor (GDNF) to treat retinal degenerative diseases. This study examined the potential clinical application of the CAC ECT gel, focusing on its safety, performance, and termination through doxycycline (Dox) administration in the eyes of healthy New Zealand White rabbits, as well as its therapeutic efficacy in rabbits with sodium-iodate (SI)-induced retinal degeneration. The findings indicated that the CAC ECT gel can be safely implanted without harming the retina or lens, displaying resistance to degradation, facilitating cell attachment, and secreting bioactive GDNF. Furthermore, the GDNF levels could be modulated by the number of implants. Moreover, Dox administration was effective in terminating gel function without causing retinal damage. Notably, rabbits with retinal degeneration treated with the gels exhibited significant functional recovery in both a-wave and b-wave amplitudes and showed remarkable efficacy in reducing photoreceptor apoptosis. Given its biocompatibility, mechanical stability, controlled drug release, terminability, and therapeutic effectiveness, our CAC ECT gel presents a promising therapeutic strategy for various retinal diseases in a clinical setting, eliminating the need for immunosuppressants.

3D cell cultures are indispensable in recapitulating in vivo environments. Among the many 3D culture methods, culturing adherent cells on hydrogel beads to form spheroid-like structures is a powerful strategy for maintaining high cell viability and functions in the adherent states. However, high-throughput, scalable technologies for 3D imaging of individual cells cultured on the hydrogel scaffolds are lacking. This study reports the development of a high throughput, scalable 3D imaging flow cytometry platform for analyzing spheroid models. This platform is realized by integrating a single objective fluorescence light-sheet microscopy with a microfluidic device that combines hydrodynamic and acoustofluidic focusing techniques. This integration enabled unprecedentedly high-throughput and scalable optofluidic 3D imaging, processing 1310 spheroids consisting of 28 117 cells min-1. The large dataset obtained enables precise quantification and comparison of the nuclear morphology of adhering and suspended cells, revealing that the adhering cells have smaller nuclei with less rounded surfaces. This platform's high throughput, robustness, and precision for analyzing the morphology of subcellular structures in 3D culture models hold promising potential for various biomedical analyses, including image-based phenotypic screening of drugs with spheroids or organoids.

Tissue engineering holds great promise for regenerative medicine, drug discovery, and as an alternative to animal models. However, as soon as the dimensions of engineered tissue exceed the diffusion limit of oxygen and nutriments, a necrotic core forms leading to irreversible damage. To overcome this constraint, the establishment of a functional perfusion network is essential. In this work, digital light processing bioprinting is used to encapsulate endothelial progenitor cells (EPCs) in 3D light-cured hydrogel scaffolds to guide them toward vascular network formation. In these scaffolds, EPCs proliferate and self-organize within a few days into branched tubular structures with predefined geometry, forming capillary-like vascular tubes or trees of diameters in the range of 10 to 100 µm. Presenting a confluent monolayer wall of cells strongly connect by tight junctions around a central lumen-like space, these structures can be microinjected with a fluorescent dye and are stable for several weeks in vitro. These endothelial structures can be recovered and manipulated in an alginate patch without altering their shape or viability. This approach opens new opportunities for future applications, such as stacking with other cell sheets or multicellular constructs to yield bioengineered tissue with higher complexity and functionality.

Ex vivo tissue engineering is an effective therapeutic approach for the treatment of severe cartilage diseases that require tissue replenishment or replacement. This strategy demands scaffolds that are durable enough for long-term cell culture to form artificial tissue. Additionally, such scaffolds must be biocompatible to prevent the transplanted matrix from taking a toll on the patient's body. From the viewpoint of structure and bio-absorbability, a β-tricalcium phosphate (β-TCP) fiber scaffold (βTFS) is expected to serve as a good scaffold for tissue engineering. However, the fragility and high solubility of β-TCP fibers make this matrix unsuitable for long-term cell culture. To solve this problem, we developed an alginate-coated β-TCP fiber scaffold (βTFS-Alg). To assess cell proliferation and differentiation in the presence of βTFS-Alg, we characterized ATDC5 cells, a chondrocyte-like cell line, when grown in this matrix. We found that alginate coated the surface of βTFS fiber and suppressed the elution of Ca2+ from β-TCP fibers. Due to the decreased solubility of βTFS-Alg compared with β-TCP, the former provided an improved scaffold for long-term cell culture. Additionally, we observed superior cell proliferation and upregulation of chondrogenesis marker genes in ATDC5 cells cultured in βTFS-Alg. These results suggest that βTFS-Alg is suitable for application in tissue culture.

Three-dimensional cell culture spheroids are commonly used for drug evaluation studies because they can produce large quantities of homogeneous cell aggregates. As the spheroids grow, nutrients supplied from outer spheroid regions render the inner spheroid areas hypoxic and hyponutrient, which makes them unobservable through confocal microscopy. In this study, we fabricated a cancer cell aggregate culture device that facilitates the observation of nutrient and oxygen gradients. An alginate gel fiber was created in the cell culture chamber to ensure a flow path for supplying the culture medium. A gradient of nutrients and oxygen was generated by positioning the flow channel close to the edge of the chamber. We devised a fabrication method that uses calcium carbonate as a source of Ca2+ for the gelation of sodium alginate, which has a slow reaction rate. We then cultured a spheroid of HCT116 cells, which were derived from human colorectal carcinoma using a fluorescent ubiquitination-based cell cycle indicator. Fluorescence observation suggested the formation of a hypoxic and hyponutrient region within an area approximately 500 µm away from the alginate gel fiber. This indicates the development of a cancer cell aggregate culture device that enables the observation of different nutrition and oxygen states.

There is a growing interest in the production of bioinks that on the one hand, are biocompatible and, on the other hand, have mechanical properties that allow for the production of stable constructs that can survive for a long time after transplantation. While the selection of the right material is crucial for bioprinting, there is another equally important issue that is currently being extensively researched-the incorporation of the vascular system into the fabricated scaffolds. Therefore, in the following manuscript, we present the results of research on bioink with unique physico-chemical and biological properties. In this article, two methods of seeding cells were tested using bioink B and seeding after bioprinting the whole model. After 2, 5, 8, or 24 h of incubation, the flow medium was used in the tested systems. At the end of the experimental trial, for each time variant, the canals were stored in formaldehyde, and immunohistochemical staining was performed to examine the presence of cells on the canal walls and roof. Cells adhered to both ways of fiber arrangement; however, a parallel bioprint with the 5 h incubation and the intermediate plating of cells resulted in better adhesion efficiency. For this test variant, the percentage of cells that adhered was at least 20% higher than in the other analyzed variants. In addition, it was for this variant that the lowest percentage of viable cells was found that were washed out of the tested model. Importantly, hematoxylin and eosin staining showed that after 8 days of culture, the cells were evenly distributed throughout the canal roof. Our study clearly shows that neovascularization-promoting cells effectively adhere to ECM-based pancreatic bioink. Summarizing the presented results, it was demonstrated that the proposed bioink compositions can be used for bioprinting bionic organs with a vascular system formed by endothelial cells and fibroblasts.

Suspension cell culture and rigid commercial substrates are the most common methods to clinically manufacture therapeutic CAR-T cells ex vivo. However, suspension culture and nano/micro-scale commercial substrates poorly mimic the microenvironment where T cells naturally develop, leading to profound impacts on cell proliferation and phenotype. To overcome this major challenge, macro-scale substrates can be used to emulate that environment with higher precision. This work employed a biocompatible thermo-responsive material with tailored mechanical properties as a potential synthetic macro-scale scaffold to support T cell encapsulation and culture. Cell viability, expansion, and phenotype changes were assessed to study the effect of two thermo-responsive hydrogel materials with stiffnesses of 0.5 and 17 kPa. Encapsulated Pan-T and CAR-T cells were able to grow and physically behave similar to the suspension control. Furthermore, matrix stiffness influenced T cell behavior. In the softer polymer, T cells had higher activation, differentiation, and maturation after encapsulation obtaining significant cell numbers. Even when terpolymer encapsulation affected the CAR-T cell viability and expansion, CAR T cells expressed favorable phenotypical profiles, which was supported with cytokines and lactate production. These results confirmed the biocompatibility of the thermo-responsive hydrogels and their feasibility as a promising 3D macro-scale scaffold for in vitro T cell expansion that could potentially be used for cell manufacturing process.

Mesenchymal stromal cells (MSCs) have displayed potential in regenerating organ function due to their anti-fibrotic, anti-inflammatory, and regenerative properties. However, there is a need for delivery systems to enhance MSC retention while maintaining their anti-fibrotic characteristics. This study investigates the feasibility of using alginate hydrogel microstrands as a cell delivery vehicle to maintain MSC viability and phenotype. To accommodate cell implantation needs, we invented a Syringe-in-Syringe approach to reproducibly fabricate microstrands in small numbers with a diameter of around 200 µm and a porous structure, which would allow for transporting nutrients to cells by diffusion. Using murine NIH 3T3 fibroblasts and primary embryonic 16 (E16) salivary mesenchyme cells as primary stromal cell models, we assessed cell viability, growth, and expression of mesenchymal and fibrotic markers in microstrands. Cell viability remained higher than 90% for both cell types. To determine cell number within the microstrands prior to in vivo implantation, we have further optimized the alamarBlue assay to measure viable cell growth in microstrands. We have shown the effect of initial cell seeding density and culture period on cell viability and growth to accommodate future stromal cell delivery and implantation. Additionally, we confirmed homeostatic phenotype maintenance for E16 mesenchyme cells in microstrands.

The study of dose-response relationships underpins analytical biosciences. Droplet microfluidics platforms can automate the generation of microreactors encapsulating varying concentrations of an assay component, providing datasets across a large chemical space in a single experiment. A classical method consists in varying the flow rate of multiple solutions co-flowing into a single microchannel (producing different volume fractions) before encapsulating the contents into water-in-oil droplets. This process can be automated through controlling the pumping elements but lacks the ability to adapt to unpredictable experimental scenarios, often requiring constant human supervision. In this paper, we introduce an image-based, closed-loop control system for assessing and adjusting volume fractions, thereby generating unsupervised, uniform concentration gradients. We trained a shallow convolutional neural network to assess the position of the laminar flow interface between two co-flowing fluids and used this model to adjust flow rates in real-time. We apply the method to generate alginate microbeads in which HEK293FT cells could grow in three dimensions. The stiffnesses ranged from 50 Pa to close to 1 kPa in Young modulus and were encoded with a fluorescent marker. We trained deep learning models based on the YOLOv4 object detector to efficiently detect both microbeads and multicellular spheroids from high-content screening images. This allowed us to map relationships between hydrogel stiffness and multicellular spheroid growth.

Chlorinated aliphatic hydrocarbons (CAHs), such as cis-1,2-dichloroethylene (cDCE), are prevalent in groundwater at many locations throughout the United States. When immobilized in hydrogel beads with slow-release compounds, the bacteria strain Rhodococcus rhodochrous ATCC 21198 can be used for the in situ bioremediation of cDCE. These hydrogel beads must exhibit high mechanical strength and resist degradation to extend the lifetime of slow-release compounds and bioremediation. We engineered poly(vinyl)-alcohol - alginate (PVA-AG) beads to immobilize ATCC 21198 with the slow-release compound, tetrabutoxysilane (TBOS) that produces 1-butanol as a growth substrate, for high mechanical strength. We optimized three inputs (concentration of PVA, concentration of AG, and the crosslinking time) on two responses (compressive modulus and rate of oxygen utilization) for batch incubation experiments between 1 and 30 days using a design of experiments approach. The predictive models generated from design of experiments were then tested by measuring the compressive strength, oxygen utilization, and abiotic rates of hydrolysis for a predicted optimal bead formulation. The result of this study generated a hydrogel bead with immobilized R. rhodochrous ATCC 21198 and TBOS that exhibited a high compressive modulus on day 1 and day 30, which was accurately predicted by models. These hydrogel beads exhibited low metabolic activity based on oxygen rates on day 1 and day 30 but were not accurately predicted by the models. In addition, the ratio between oxygen utilization and abiotic rates of hydrolysis were observed to be roughly half of what was expected stoichiometrically. Lastly, we demonstrated the capability to use these beads as a bioremediation technology for cDCE as we found that, for all bead formulations, cDCE was significantly reduced after 30 days. Altogether, this work demonstrates the capability to capture and enhance the material properties of the complex hydrogel beads with predictive models yet signals the need for more robust methods to understand the metabolic activity that occurs in the hydrogel beads.

Chemotherapy is a conventional treatment that uses drugs to kill cancer cells; however, it may induce side effects and may be incompletely effective, leading to the risk of tumor recurrence. To address this issue, we developed novel injectable thermal/near-infrared (NIR)-responsive hydrogels to control drug release. The injectable hydrogel formulation was composed of biocompatible alginates, poly(N-acryloyl glycinamide) (PNAGA) copolymers with an upper critical solution temperature, and NIR-responsive cross-linkers containing coumarin groups, which were gelated through bioorthogonal inverse electron demand Diels-Alder reactions. The hydrogels exhibited quick gelation times (120-800 s) and high drug loading efficiencies (>90%). The hydrogels demonstrated a higher percentage of drug release at 37 °C than that at 25 °C due to the enhanced swelling behavior of temperature-responsive PNAGA moieties. Upon NIR irradiation, the hydrogels released most of the entrapped doxorubicin (DOX) (97%) owing to the cleavage of NIR-sensitive coumarin ester groups. The hydrogels displayed biocompatibility with normal cells, while induced antitumor activity toward cancer cells. DOX/hydrogels treated with NIR light inhibited tumor growth in nude mice bearing tumors. In addition, the injected hydrogels emitted red fluorescence upon excitation at a green wavelength, so that the drug delivery and hydrogel degradation in vivo could be tracked in the xenograft model.

Glandular cancers are amongst the most prevalent types of cancer, which can develop in many different organs, presenting challenges in their detection as well as high treatment variability and failure rates. For that purpose, anticancer drugs are commonly tested in cancer cell lines grown in 2D tissue culture on plastic dishesin vitro, or in animal modelsin vivo. However, 2D culture models diverge significantly from the 3D characteristics of living tissues and animal models require extensive animal use and time. Glandular cancers, such as prostate cancer-the second leading cause of male cancer death-typically exist in co-centrical architectures where a cell layer surrounds an acellular lumen. Herein, this spatial cellular position and 3D architecture, containing dual compartments with different hydrogel materials, is engineered using a simple co-axial nozzle setup, in a single step utilizing prostate as a model of glandular cancer. The resulting hydrogel soft structures support viable prostate cancer cells of different cell lines and enable over-time maturation into cancer-mimicking aggregates surrounding the acellular core. The biofabricated cancer mimicking structures are then used as a model to predict the inhibitory efficacy of the poly ADP ribose polymerase inhibitor, Talazoparib, and the antiandrogen drug, Enzalutamide, in the growth of the cancer cell layer. Our results show that the obtained hydrogel constructs can be adapted to quickly obtain 3D cancer models which combine 3D physiological architectures with high-throughput screening to detect and optimize anti-cancer drugs in prostate and potentially other glandular cancer types.

Numerous studies have explored the cultivation of muscle cells using non-animal materials for cultivated meat production. Achieving muscle cell proliferation and alignment using 3D scaffolds made from plant-based materials remains challenging. This study introduces a technique to culture and align muscle cells using only plant-based materials, avoiding toxic chemical modifications. Zein-alginate fibers (ZA fibers) were fabricated by coating zein protein onto alginate fibers (A fibers). Zein's excellent cell compatibility and biodegradability enable high cell adhesion and proliferation rates, and the good ductility of the ZA fibers enable a high strain rate (>75%). We demonstrate mature and aligned myotube formation in ZA fibers, providing a simple way to align muscle cells using plant-based materials. Additionally, cultivated meat was constructed by assembling muscle, fat, and vessel fibers. This method holds promise for the future mass production of cultivated meat.

The manufacturing of 3D cell scaffoldings provides advantages for modeling diseases and injuries as it enables the creation of physiologically relevant platforms. A triple-flow microfluidic device is developed to rapidly fabricate alginate/graphene hollow microfibers based on the gelation of alginate induced with CaCl2 . This five-channel microdevice actualizes continuous mild fabrication of hollow fibers under an optimized flow rate ratio of 300:200:100 µL min-1 . The polymer solution is 2.5% alginate in 0.1% graphene and a 30% polyethylene glycol solution is used as the sheath and core solutions. The biocompatibility of these conductive microfibers by encapsulating mouse astrocyte cells (C8D1A) within the scaffolds is investigated. The cells can successfully survive both the manufacturing process and prolonged encapsulation for up to 8 days, where there is between 18-53% of live cells on both the alginate microfibers and alginate/graphene microfibers. These unique 3D hollow scaffolds can significantly enhance the available surface area for nutrient transport to the cells. In addition, these conductive hollow scaffolds illustrate unique advantages such as 0.728 cm3  gr-1 porosity and two times more electrical conductivity in comparison to alginate scaffolds. The results confirm the potential of these scaffolds as a microenvironment that supports cell growth.

Chimeric antigen receptor (CAR) T cells exhibit promising progress in addressing hematologic malignancies. However, CAR-T therapy for solid tumors remains limited, with no FDA-approved CAR-T products available for clinical use at present. Primary reasons include insufficient infiltration, accumulation, tumor immunosuppression of the microenvironment, and related side effects. Single utilization of CAR-T cannot effectively overcome these unfavorable obstacles. A probable effective pathway to achieve a better CAR-T therapy effect would be to combine the benefits of biomaterials-based technology. In this article, comprehensive biomaterials strategies to break through these obstacles of CAR-T cell therapy at the tumor sites are summarized, encompassing the following aspects: 1) generating orthotopic CAR-T cells; 2) facilitating CAR-T cell trafficking; 3) stimulating CAR-T cell expansion and infiltration; 4) improving CAR-T cell activity and persistence; 5) reprogramming the immunosuppressive microenvironments. Additionally, future requirements for the development of this field, with a specific emphasis on promoting innovation and facilitating clinical translation, are thoroughly discussed.

The periodontal ligament (PDL) is a distinctive yet critical connective tissue vital for maintaining the integrity and functionality of tooth-supporting structures. However, PDL repair poses significant challenges due to the complexity of its mechanical microenvironment encompassing hard-soft-hard tissues, with the viscoelastic properties of the PDL being of particular interest. This review delves into the significant role of viscoelastic hydrogels in PDL regeneration, underscoring their utility in simulating biomimetic three-dimensional microenvironments. We review the intricate relationship between PDL and viscoelastic mechanical properties, emphasizing the role of tissue viscoelasticity in maintaining mechanical functionality. Moreover, we summarize the techniques for characterizing PDL's viscoelastic behavior. From a chemical bonding perspective, we explore various crosslinking methods and characteristics of viscoelastic hydrogels, along with engineering strategies to construct viscoelastic cell microenvironments. We present a detailed analysis of the influence of the viscoelastic microenvironment on cellular mechanobiological behavior and fate. Furthermore, we review the applications of diverse viscoelastic hydrogels in PDL repair and address current challenges in the field of viscoelastic tissue repair. Lastly, we propose future directions for the development of innovative hydrogels that will facilitate not only PDL but also systemic ligament tissue repair. STATEMENT OF SIGNIFICANCE.

Three-dimensional (3D) cell cultures have emerged as valuable tools in cancer research, offering significant advantages over traditional two-dimensional (2D) cell culture systems. In 3D cell cultures, cancer cells are grown in an environment that more closely mimics the 3D architecture and complexity of in vivo tumors. This approach has revolutionized cancer research by providing a more accurate representation of the tumor microenvironment (TME) and enabling the study of tumor behavior and response to therapies in a more physiologically relevant context. One of the key benefits of 3D cell culture in cancer research is the ability to recapitulate the complex interactions between cancer cells and their surrounding stroma. Tumors consist not only of cancer cells but also various other cell types, including stromal cells, immune cells, and blood vessels. These models bridge traditional 2D cell cultures and animal models, offering a cost-effective, scalable, and ethical alternative for preclinical research. As the field advances, 3D cell cultures are poised to play a pivotal role in understanding cancer biology and accelerating the development of effective anticancer therapies. This review article highlights the key advantages of 3D cell cultures, progress in the most common scaffold-based culturing techniques, pertinent literature on their applications in cancer research, and the ongoing challenges.

A tumor is a complex cluster with many types of cells in the microenvironment that help it grow. Macrophages, immune cells whose main role is to maintain body homeostasis, represent in the majority of cancers the most important cell population. In this context, they are called tumor-associated macrophages (TAMs) because of their phenotype, which contributes to tumor growth. In order to limit the use of animals, there is a real demand for the creation of in vitro models able to represent more specifically the complexity of the tumor microenvironment (TME) in order to characterize it and evaluate new treatments. However, the two-dimensional (2D) culture, which has been used for a long time, has shown many limitations, especially in terms of tumor representation. The three-dimensional (3D) models, developed over the last 20 years, have made it possible to get closer to what happens in vivo in terms of phenotypic and functional characteristics. Due to their architectural similarity to in vivo tissues, they provide a more physiologically relevant in vitro system. Most recently, it is the development of 3D coculture models in which two or three cell lines are cultured together that has allowed a better representation of TME with cell-cell interactions. Unfortunately, there is no clear direction for the design of these models at this time. In this Review on the coculture between cancer cells and TAMs, an in-depth analysis is performed to answer multiple questions on the conception of these models: Which models to use, and with which material and cancer lineage? Which monocyte or macrophage lines should be added to the coculture? And how can these models be exploited?

Bone morphogenetic protein-2 (BMP-2) has been commercially approved by the Food and Drug Administration for use in bone defects and diseases. BMP-2 promotes osteogenic differentiation of mesenchymal stem cells. In bone tissue engineering, BMP-2 incorporated into scaffolds can be used for stimulating bone regeneration in organoid construction, drug testing platforms, and bone transplants. However, the high dosage and uncontrollable release rate of BMP-2 challenge its clinical application, mainly due to the short circulation half-life of BMP-2, microbial contamination in bone extracellular matrix hydrogel, and the delivery method. Moreover, in clinical translation, the requirement of high doses of BMP-2 for efficacy poses challenges in cost and safety. Based on these, novel strategies should ensure that BMP-2 is delivered precisely to the desired location within the body, regulating the timing of BMP-2 release to coincide with the bone healing process, as well as release BMP-2 in a controlled manner to optimize its therapeutic effect and minimize side effects. This review highlights improvements in bone tissue engineering applying spatiotemporal and controlled BMP-2 delivery, including molecular engineering, biomaterial modification, and synergistic therapy, aiming to provide references for future research and clinical trials.

Digestive system tumors are the leading cause of cancer-related deaths worldwide. Despite ongoing research, our understanding of their mechanisms and treatment remain inadequate. One promising tool for clinical applications is the use of gastrointestinal tract tumor organoids, which serve as an important in vitro model. Tumor organoids exhibit a genotype similar to the patient's tumor and effectively mimic various biological processes, including tissue renewal, stem cell, and ecological niche functions, and tissue response to drugs, mutations, or injury. As such, they are valuable for drug screening, developing novel drugs, assessing patient outcomes, and supporting immunotherapy. In addition, innovative materials and techniques can be used to optimize tumor organoid culture systems. Several applications of digestive system tumor organoids have been described and have shown promising results in related aspects. In this review, we discuss the current progress, limitations, and prospects of this model for digestive system tumors.