Investigation of toxicological profile and possible side effects of engineered nanomaterials (ENMs) is of high importance. Historically, two-dimensional (2D) cell culture was used to study the toxicity of the ENMs, but due to their inability to simulate in vivo cell behavior, three-dimensional (3D) cell culture systems have been developed. Nanotoxicity studies initiate with in vitro experiments and continue with in vivo studies, which are very challenging and sometimes accompanied by conflicting data due to the in vitro-in vivo gap. Thus, scientists are turning their attention to microfabrication techniques and engineered systems "called organ-on-a-chips", which act as an intermediate between in vivo and in vitro systems. The present account tries to review the classical study models and suitably cover the emerging 3D culture models including scaffold-free and scaffold-based 3D cell cultures, 3D co-culture with direct contact and without cell-cell contact methods as well as microfluidic-based tissue chips and organoids. Overall, this review aims to give readers a better insight about the ENMs' toxicology and fill the gaps between the knowledge and practical techniques. Hopefully, the presented information will resolve the issues of 2D in vitro cultures and display the clinically relevant responses to the concerns of therapeutic ENMs.

Three-dimensional cell culture in vitro could better recapitulate the biological behavior of cells in vivo.

Alteration in gene expression patterns and increased drug resistance in MCF7 breast cancer cells cultured on 3D collagen-based scaffolds.

Mumio and bladder cancer: unlocking its potential in 3D cell culture.

ABSTRACT Objective: Cancer, characterized by uncontrolled cell proliferation and invasion into surrounding tissues, is a leading cause of global mortality. Traditional two-dimensional (2D) cell culture systems fail to adequately replicate the tumor microenvironment (TME). In contrast, three-dimensional (3D) culture systems, which better simulate cell–cell and cell–extracellular matrix (ECM) interactions, have become powerful tools in biomedical research. This study aims to compare the spheroid formation capacity of A549 lung cancer cells using three different 3D culture methods: ultra-low attachment (ULA) plates, agarose hydrogel, and the hanging drop technique. The primary objective is to identify the most effective spheroid formation method for A549 cells and to provide findings that can guide future biomedical research, particularly in cancer modeling, drug screening studies, and investigations of the tumor microenvironment.Materials and Methods: A549 cells were cultured using three different 3D culture methods: ultra-low attachment plates, agarose hydrogel, and the hanging drop method. In the ultra-low attachment method, spheroid formation was observed at cell densities of 5,000, 10,000, and 30,000 cells/ml. In the agarose hydrogel method, agarose concentrations of 1%, 1.5%, and 2% were used to evaluate cell aggregation and spheroid stability. In the hanging drop method, cells aggregated under the influence of gravity. Spheroid diameter and area were analyzed using ImageJ software.Results: In this study, the spheroid formation capacity of A549 lung cancer cells was evaluated using three different three-dimensional (3D) culture methods. The ultra-low attachment (ULA) plate method allowed cell aggregation; however, the resulting structures were not large or compact enough to be classified as spheroids. The hanging drop method showed that cells formed small clusters by day 3 but failed to develop a compact and stable spheroid structure by day 7. The agarose hydrogel method, particularly at a 2% agarose concentration, demonstrated the highest spheroid formation capacity compared to the other methods. In this method, spheroid formation began at 72 hours depending on cell density, with significant growth observed at a density of 30,000 cells/ml (p < 0.0001). Trypan Blue staining results indicated that 2% agarose and cell densities of 10,000–30,000 cells/ml provided the highest cell viability. Specifically, 4,800 viable cells were counted at a density of 30,000 cells/ml, while 3,600 viable cells were observed at 10,000 cells/ml. These findings suggest that the agarose hydrogel method, especially at 2% agarose concentration and higher cell densities, offers optimal spheroid formation and cell viability for A549 lung cancer cells.Conclusion: This study demonstrated that the agarose hydrogel method effectively promoted stable and organized spheroid formation in A549 lung cancer cells. Notably, the 2% agarose concentration was identified as the most effective condition for maintaining cell viability and optimizing spheroid size. In contrast, the ultra-low attachment (ULA) plate and hanging drop methods exhibited limited spheroid formation capacity, resulting in less compact and disorganized structures. These findings emphasize the critical role of three-dimensional (3D) cell culture methods in biomedical research, particularly for experimental tumor modeling and drug screening studies. In this context, the agarose hydrogel method, with its high spheroid formation capacity and ability to support cell viability, emerges as a promising 3D culture model that warrants further exploration in cancer research.

Adipocyte spheroids offer a promising three-dimensional (3D) cell culture model for obesity research, as they reproduce the structure and cell-cell interaction of adipose tissue more accurately compared to two-dimensional (2D) cultures. However, the mass production of uniform, small adipocyte spheroids remains challenging, limiting their use in large-scale analyses, such as drug screening. Here, we develop a method that combines simple microfluidics with templated emulsification to enable the large-scale production of small, uniform adipocyte spheroids. By encapsulating preadipocytes in hollow agarose microcapsules and incubating them for 2 days, we reproducibly generated more than 100,000 uniform spheroids with diameters of approximately 60 μm. These preadipocytes were subsequently differentiated into adipocyte spheroids through an 8-day induction period. This platform facilitates large-scale 3D analysis for obesity research and can be adapted to produce various other spheroid and organoid models, broadening its utility in biomedical research.

Biological research groups may face a high barrier to entry when constructing custom 3D cell culture devices to investigate multi-tissue interactions in vitro. Standard fabrication methods such as lithography, etching, or molding are expensive and require specialized equipment and expertise. To address this, we developed an accessible approach for producing polyethylene glycol (PEG)-based cell culture devices using stereolithography 3D printing with a polydimethylsiloxane intermediate mold. Both the intermediate molding steps and the sterilized final device show low cytotoxicity, and the final device swells to predictable dimensions and retains its shape for at least 10 days. We used this approach to develop a human pluripotent stem cell-derived neural spheroid outgrowth model that supports directed neurite extension over 14 days. Together, this method provides a highly customizable, affordable platform for rapid fabrication of PEG-based microphysiological systems for diverse tissue engineering applications.

Exercise is well known to promote tendon healing, an effect traditionally attributed to mechanical loading-induced responses within the tendon itself. However, skeletal muscle also functions as a secretory organ, releasing bioactive factors (secretome) during exercise that influence various tissues. We hypothesized that muscle-derived secretome released during exercise may also contribute to tendon healing. To test this, we applied mechanical loading to cultured muscle cells (myoblasts) using the FlexCell tension system to simulate exercise invitro. Our previous studies, using 2D-cultured tendon cells (tenocytes), have demonstrated that secretome from statically loaded myoblasts, particularly under 2% loading, enhanced tendon healing-related responses. Building upon these findings, we employed a 3D tendon construct model to more closely mimic invivo healing conditions. We found that secretome derived from statically loaded myoblasts, especially at 2% loading, promoted tendon healing-related processes as compared with the control group, which received no secretome treatment (no conditioned media). These included increased cell-covered area, expression of the tenocyte marker scleraxis (SCX), and elevated production of Type I and III collagens at an early stage (Day 7). Additionally, a reduction in type III collagen production was found at a later stage (Day 14), suggesting a potentially accelerated healing process. These findings highlight the therapeutic potential of the muscle-derived secretome in promoting tendon healing and may inform future strategies for rehabilitation and regenerative medicine.

Hydrogels restrict protein transport to different extents, with nanoporous synthetic polymer networks providing far less protein permeability compared to microporous biopolymer networks. To evaluate whether reduced permeability was a driving factor in reduced cell viability in synthetic hydrogels, we compared poly(ethylene glycol) vinyl sulfone (PEG-VS) hydrogels with Matrigel to quantify the influences of modulus, transport, and confinement on encapsulated cells. We observed extensive reductions in cell viability when encapsulated in PEG-VS gels compared to Matrigel. In transwell experiments that decouple hydrogel-restricted serum from cell-gel adhesion, serum restriction reduced cell viability, matching the cell viability observed in 3D cultures. Our unique combination of 2D and 3D hydrogel-based cell cultures provides a framework for investigating the intersecting effects of the cell microenvironment's properties on cell viability. This work demonstrates that biomaterial-restricted protein transport is a critical design consideration when using synthetic 3D cell culture hydrogels.

The process of developing a drug is complex and involves many steps, from basic research (bench) to patient applications (bedside), which are conducted to ensure the drug is both safe and effective. In cancer research, the failure rate is high when translating basic findings to clinical trials. One of the main factors probably contributing to high failure rates is the basic quality of in vitro and in vivo study designs. Advanced basic cancer research techniques, including various types of 3D cell culture, the use of valuable organoids, organs, or tumors on chips, traditional or automated Western blots, omics research, advanced imaging techniques, usage of cutting-edge preclinical models and others, may produce inaccurate results for translational research if the basic study design is not carefully planned, especially when drugs or compounds are involved. In this manuscript, the author discussed (i) the importance of understanding and applying pharmacokinetic data in basic research, (ii) a proper comparison of the efficacy and safety of investigational drugs with the standard of care, (iii) the importance of following the actual route of drug administration as experienced by patients, the cruciality of human-to-animal dose conversion, and dose frequencies in animal models, (iv) significance of the age, gender, and strain of mice, along with adherence to the ARRIVE guidelines for ensuring transparency in conducting and reporting preclinical research, (v) benefits of having both subcutaneous and metastasis models in preclinical studies, (vi) the impact of comorbidities and related cancer drug studies in animal models and (vii) the importance of testing drug candidate/s in model mimicking acidic tumor microenvironment.

A workflow for quantifying cell quiescence in 3D spheroids.

Functionalized peptide hydrogel to generate human insulin-producing cells in vitro.

The 3Rs are guiding principles that must be followed when designing studies and conducting toxicity research. There is ongoing controversy regarding the use of animals in research. Moreover, the European Union outlawed animal experimentation in cosmetic products on 11 March 2013. More recently, the FDA Modernization Act 2.0 removed the requirement to use animal studies as part of the process for obtaining a licence for a biological product. These moral, ethical, and legal constraints have increased the need for alternative testing procedures. 3D cell cultures have gained popularity in recent years. Small microfluidic platforms known as "organs-on-chips" are dynamic cell cultures that mimic specific microenvironments. These microchips allow scientists to collect in vivo-like data. Organs-on-chips are considered a promising replacement for animal testing, and toxicity research is rapidly adopting this novel approach, much like other scientific fields, such as neuroscience, stem cell research, and cancer investigations. Therefore, the purpose of this review is to discuss the areas of toxicology where these platforms are currently being used, summarise the most recent toxicological applications of the aforementioned platforms, discuss the opportunities and challenges they present for toxicological research, and explore the interdisciplinary approaches applied within the field of toxicology.

Branching morphogenesis is a key process for constructing the tree-like architecture of multiple organs. The mechanisms regulating pancreatic ductal morphogenesis are still poorly understood, especially in the context of the particular pH dynamics of this organ. Indeed, ductal cells periodically release an alkaline juice to balance stomach acidity during digestion. This leads to a drop in extracellular pH (pHe) in the extracellular matrix (ECM) to maintain intracellular pH (pHi) homeostasis. Among the transporters involved in pH regulation, NHE1 also regulates epithelial branching morphogenesis in various tissues/organs. However, neither the effect of the changing pHe nor the role of NHE1 in branching morphogenesis has been investigated in a physiomimetic model in the human pancreas. Here, using 3D organotypic cultures of human pancreatic ductal cells (HPDE), we found that cells seeded on a Matrigel rich-ECM resembling normal ECM formed branched duct-like structures, which did not form on a more fibrotic Collagen I-rich ECM. Further, these cells overexpressed NHE1 mainly at the basolateral membrane. Ductal morphogenesis was affected by acidic pHe (pHe 6.7), which determined a hyper-branched network, and this was further increased by the inhibition of NHE1. We conclude that ECM composition and extracellular acidosis modulate branching morphogenesis in pancreatic ductal HPDE cells via NHE1 activity.

A gravity-driven millifluidic platform with unidirectional flow for 3D cell culture and drug screening applications

The development of targeted radionuclide- and chemotherapeutics-based drugs requires in vitro models that reflect the structural and molecular complexity of solid tumors. Here, we present a high-throughput 3D cell culture platform for targeted drug response studies. The platform was based on agarose micro-dishes that generate 81 tumor spheroids. The platform was validated across eight human and murine cancer cell lines that overexpress receptors of the HER family. The high spheroid yield supports robust quantitative analysis of binding of radiolabeled compounds, while spheroid trapping in microwells enables medium exchange for precise drug exposure. Therefore, we evaluated targeted radionuclide therapy of EMT-HER2 spheroids using the HER2-specific affibody PEP48937 labelled with terbium-161. We demonstrated both HER2-specific binding to spheroids and receptor-specific therapeutic effects, including reduced spheroid proliferation over time and impaired cell migration. These results highlight the platform potential to accelerate the development of targeted cancer therapeutics for biomedicine.

ABSTRACT Three‐dimensional (3D) multicellular models are considered ideal methods for bridging the gap between two‐dimensional (2D) cell culture and animal models, which are widely used in organogenesis, disease models, drug development, and regenerative medicine. Cell culture technologies determine the physical and biological properties of multicellular spheroids or organoids that affect the authenticity, stability, assessment, and throughput of the 3D multicellular system. Micro patterns, characterized as a coating of specific adhesion matrices on substrates, can control cell behaviors and fate by limiting the space available for cell spreading. micro patterns are used to culture non‐tumor or tumor spheroids and organoids with effective control of size and arrangement, which is suitable for large‐scale and standardized culture to generate 3D multicellular models. This comprehensive review summarizes the advantages and applications of 3D multicellular models and discusses the characteristics of general 3D cell culture technologies. We discuss the basic applications of micro pattern technologies and highlight the specific advantages and features of micropattern (as a 3D cell culture platform) in non‐tumor research (regenerative medicine, developmental biology, disease modelling, and monoclonal cell culture) and tumor research (tumor microenvironment (TME) and drug screening). Finally, the fabrication of micro patterns (bio inks, fabrication methods for micro patterns, morphology, and quality of micro patterns) is described.

We established a 3D model of human NT2/D1-derived early neural progenitor cells immobilised in alginate microfibres as a system for testing the neurotoxicity of energy drinks and their components, either alone, together, or in combination with alcohol. The in vitro system supports the retinoic acid-induced neurogenesis of NT2/D1 cells, and the proliferative capacity of the NT2/D1-derived early neural progenitor cells was maintained in the 3D environment. Cell cycle distribution and the expression of pluripotency markers (SOX2, OCT4 and NANOG), early neural markers (SOX3, PAX6 and miR-219), and Cyclin D1 (a marker of neural commitment), showed profiles characteristic of early neural progenitors. Treatments with an energy drink and its major components (caffeine and taurine) - either alone, together, or in combination with alcohol - had different effects on the proliferative capacity of the NT2/D1-derived early neural progenitor cells in the 2D and 3D models. In the 2D-cultured cells, all treatments except for caffeine led to a significant decrease, while cells within the 3D model exhibited a significant increase after treatment with caffeine, or after combined treatment with energy drink and alcohol. Preliminary findings suggesting that there were no treatment effects on OCT4 and PAX6 protein expression in either model, should be further confirmed. This human cell-based 3D model could potentially represent a rapid and cost-effective system for assessing the acute and long-term neurotoxicity of various compounds.

Tumor spheroids, the most widely used model of 3D cell culture, have emerged as a viable platform for assessing drug responses. However, high-throughput validation of novel drugs using tumor spheroids remains hindered by the challenges in generating large-scale, homogeneous, and functionally relevant spheroids. Here, a flow-focusing droplet microfluidic platform is developed for high-throughput generation of uniform tumor spheroids, producing over 50000 droplets within 5 min, with each microdroplet serving as an individual bioreactor for spheroid formation. The initial size of the tumor spheroids is tuned based on cell concentration and water-to-oil flow rate ratio during microdroplet generation. After being released from the microdroplets, the 3D tumor spheroids continue growing, reaching diameters exceeding 300 µm. The growth and functional characteristics of the spheroids are examined both in a liquid environment and in a 3D collagen matrix. Moreover, these tumor spheroids enable assessment of the therapeutic efficacy of siRNA-based nanomedicine that demonstrates enhanced performance compared to free siRNA treatments. This platform offers a robust and scalable approach for evaluating novel nanomedicines, providing valuable insights into their therapeutic potential and underlying mechanisms of action.

Engineering biocompatible polylactic acid (PLA) microcarriers for enhanced 3D cell culture.