The progression of breast cancer is influenced by the stiffness of the extracellular matrix (ECM), which becomes stiffer as cancer advances due to increased collagen IV and laminin secretion by cancer-associated fibroblasts. Intriguingly, breast cancer cells cultivated in two-dimensions exhibit a less aggressive behavior when exposed to weightlessness, or microgravity conditions. This study aims to elucidate the interplay between matrix stiffness and microgravity on breast cancer progression. For this purpose, three-dimensional spheroids of breast cancer cell lines (MCF-7 and MDA-MB-231) were formed. These spheroids were subsequently bioprinted in hydrogels of varying stiffness, obtained by the mixing of gelatin methacrylate and poly(ethylene) glycol diacrylate mixed at different ratios. The constructs were printed with a custom stereolithography (SLA) bioprinter converted from a low-cost, commercially available 3D printer. These bioprinted structures, encapsulating breast cancer spheroids, were then placed in a clinostat (microgravity simulation device) for a duration of seven days. Comparative analyses were conducted between objects cultured under microgravity and standard earth gravity conditions. Protein expression was characterized through fluorescent microscopy, while gene expression of MCF-7 constructs was analyzed via RNA sequencing. Remarkably, the influence of a stiffer ECM on the protein and gene expression levels of breast cancer cells could be modulated and sometimes even reversed in microgravity conditions. The study's findings hold implications for refining therapeutic strategies for advanced breast cancer stages - an array of genes involved in reversing aggressive or even metastatic behavior might lead to the discovery of new compounds that could be used in a clinical setting.

Recent advancements in three-dimensional (3D) cell culture technologies, such as cell spheroids, organoids, and 3D bioprinted tissue constructs, have significantly improved the physiological relevance of in vitro models. These models better mimic tissue structure and function, closely emulating in vivo characteristics and enhancing phenotypic analysis, critical for basic research and drug screening in personalized cancer therapy. Despite their potential, current 3D cell culture platforms face technical challenges, which include user-unfriendliness in long-term dynamic cell culture, incompatibility with rapid cell encapsulation in biomimetic hydrogels, and low throughput for compound screening. To address these issues, we developed a 144-pillar plate with sidewalls and slits (144PillarPlate) and a complementary 144-perfusion plate with perfusion wells and reservoirs (144PerfusionPlate) for dynamic 3D cell culture and predictive compound screening. To accelerate biomimetic tissue formation, small Hep3B liver tumor spheroids suspended in alginate were printed and encapsulated on the 144PillarPlate rapidly by using microsolenoid valve-driven 3D bioprinting technology. The microarray bioprinting technology enabled precise and rapid loading of small spheroids in alginate on the pillar plate, facilitating reproducible and scalable formation of large tumor spheroids with minimal manual intervention. The bioprinted Hep3B spheroids on the 144PillarPlate were dynamically cultured in the 144PerfusionPlate and tested with anticancer drugs to measure drug effectiveness and determine the concentration required to inhibit 50% of the cell viability (IC50 value). The perfusion plate enabled the convenient dynamic culture of tumor spheroids and facilitated the dynamic testing of anticancer drugs with increased sensitivity. It is envisioned that the integration of microarray bioprinting of tumor spheroids onto the pillar plate, along with dynamic 3D cell culture in the perfusion plate, could more accurately replicate tumor microenvironments. This advancement has the potential to enhance the predictive drug screening process in personalized cancer therapy significantly.

Compared to cultured 2D cell monolayers, 3D multicellular spheroids are more realistic tumor models. Nonetheless, spheroids remain under-utilized in preclinical research, in part, because there is a lack of fluorescence sensors that can noninvasively interrogate all the individual cells within a spheroid. This present study describes a deep-red fluorogenic molecular probe for microscopic imaging of cells that contain a high level of nitroreductase enzyme activity as a biomarker of cell hypoxia. A first-generation version of the probe produced "turn-on" fluorescence in a 2D cell monolayer under hypoxic conditions; however, it was not useful in a 3D multicellular tumor spheroid because it only accumulated in the peripheral cells. To guide the probe structural optimization process, an intuitive theoretical membrane partition model was conceived to predict how a dosed probe will distribute within a 3D spheroid. The model identifies three limiting molecular diffusion pathways that are determined by a probe's membrane partition properties. A lipophilic probe with high membrane affinity rapidly becomes trapped in the membranes of the peripheral cells. In contrast, a very hydrophilic probe molecule with negligible membrane affinity diffuses rapidly through the spheroid intercellular space and rarely enters the cells. However, a probe molecule with intermediate membrane affinity undergoes sequential diffusion in and out of cells and distributes to all the cells within a spheroid. Using the model as a predictive tool, a second-generation fluorescent probe was prepared with a smaller and more hydrophilic molecular structure, and optical sectioning using structured illumination or light sheet microscopy revealed roughly even probe diffusion throughout a tumor spheroid. The membrane permeation model is likely to be broadly applicable for the structural optimization of various classes of molecules and nanoparticles to enable even distribution within a tumor spheroid.

Recent advances in cell culturing and DNA sequencing have dramatically altered the field of human microbiome research. Three-dimensional (3D) cell culture is an important tool in cell biology, in cancer research, and for studying host-microbe interactions, as it mimics the in vivo characteristics of the host environment in an in vitro system, providing reliable and reproducible models. This work provides an overview of the main 3D culture techniques applied to study interactions between host cells and pathogenic microorganisms, how these systems can be integrated with high-throughput molecular methods, and how multi-species model systems may pave the way forward to pinpoint interactions among host, beneficial microbes and pathogens.

Primary hepatocytes (PHs) are indispensable for studying liver function, drug screening, and regenerative medicine. However, freshly isolated PHs only survive for a few hours in non-adherent suspension culture. This study proposes treatment with PEG-GRGDS, a polymer-peptide conjugate comprising polyethylene glycol (PEG) and the pentapeptide sequence Gly-Arg-Gly-Asp-Ser (GRGDS), to sustain the viability of dispersed single PHs under non-adherent conditions. As a proof of concept, PHs treated with the PEG-GRGDS molecule were cultured in a microarray with single-cell-sized microwells. After 24 h of culture, enhanced cell survival was confirmed via esterase activity alongside activity for Cytochrome P450 1A1 (CYP1A1). Some liver-specific functionalities, including albumin secretion, were observed in the treated PHs. Additionally, it was observed that the length of the PEG-chain in the conjugates influenced the maintenance of single-cell dispersion and the levels of polymerized actin in the cells. These findings suggest that treatment with a polymer-peptide like PEG-GRGDS might provide a promising platform for the short-term culture of non-adherent single PHs.

The online version contains supplementary material available at 10.1007/s10616-024-00696-1.

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.

Metabolic dysfunction-associated steatohepatitis (MASH) is a leading cause of chronic liver disease with few therapeutic options. To narrow the translational gap in the development of pharmacological MASH treatments, a 3D liver model from primary human hepatocytes and non-parenchymal cells derived from patients with histologically confirmed MASH was established. The model closely mirrors disease-relevant endpoints, such as steatosis, inflammation and fibrosis, and multi-omics analyses show excellent alignment with biopsy data from 306 MASH patients and 77 controls. By combining high-content imaging with scalable biochemical assays and chemogenomic screening, multiple novel targets with anti-steatotic, anti-inflammatory, and anti-fibrotic effects are identified. Among these, activation of the muscarinic M1 receptor (CHRM1) and inhibition of the TRPM8 cation channel result in strong anti-fibrotic effects, which are confirmed using orthogonal genetic assays. Strikingly, using biosensors based on bioluminescence resonance energy transfer, a functional interaction along a novel MASH signaling axis in which CHRM1 inhibits TRPM8 via Gq/11 and phospholipase C-mediated depletion of phosphatidylinositol 4,5-bisphosphate can be demonstrated. Combined, this study presents the first patient-derived 3D MASH model, identifies a novel signaling module with anti-fibrotic effects, and highlights the potential of organotypic culture systems for phenotype-based chemogenomic drug target identification at scale.

Enzalutamide (ENZ) has emerged as a major treatment advance in castration-resistant prostate cancer (CRPC) patients; however the development of resistance remains a key challenge. The extracellular vesicles (VEs)-derived miRNAs play crucial roles tumor microenvironment cell communication, thereby influencing resistance mechanisms. Considering the urgent need for molecular biomarkers to monitor ENZ response and predict resistance, we intend to identify an EV-derived miRNA profile associated with ENZ resistance using an innovative 3D-spheroid in vitro model. Through the generation of this model, we provide a comprehensive platform for elucidating the molecular alterations involved in the process. An in vitro model of ENZ resistance was established through continuous exposure of LNCaP to increasing ENZ concentrations. A screening of 799 miRNAs from resistant and normal LNCaP cells were quantified. A bioinformatic analysis was performed using miRTarbase and Cytoscape and top 5 overexpressed miRNAs were selected, that will be analyzed in extracellular vesicles derived from ENZ resistance 3D spheroid models. We identified 12 up- and 13 downregulated miRNAs in LNCaP 30 μM ENZ cells compare to LNCaP·In silico analysis led to the construction of a 76 proteins cluster and functional enrichment revealed terms like PI3K/AKT, TFG-β and FOXO. hsa-miR-22-3p was significantly decreased at 5 and 20 μM ENZ concentration intracellularly, but significantly increased at 20 μM ENZ in EVs. hsa-miR-221-3p and miR-222-3p were upregulated in all concentrations both intracellularly and in EVs. The developed 3D-spheroid model effectively replicated the ENZ resistance to ENZ in an AR-independent manner, underscoring the importance of EVs-derived miRNAs in this adaptive process.

Cancer cell monolayers are commonly used for preclinical drug screening. However, monolayers do not begin to mimic the complexity of the tumor microenvironment, including hypoxia and nutrient gradients within the tumor. To more accurately mimic solid tumors, we developed and drug-tested an anaplastic lymphoma kinase (ALK)-positive (H3122) non-small-cell lung cancer 3D (three-dimensional) culture model using light-activated gelatin methacryloyl hydrogels. We previously demonstrated that the combination of alectinib, an ALK inhibitor, and SHP099, an SHP2 inhibitor, had synergistic efficacy in ALK-positive cell monolayers. We aimed to test this drug combination in our novel ALK-positive 3D cancer model. We first validated the 3D cultures by comparing the distribution of terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL)-positive cells in the 3D cultures with sections from time-matched mouse xenografts, finding a comparable percentage of TUNEL-positive cells in the 3D culture and xenograft inner cores at each time point. When we investigated the effect of the combination of alectinib and SHP099 in these novel 3D cultures, we found a comparable cellular response compared with our two-dimensional experiments especially with the drugs in combination. We suggest that 3D cultures be used as preclinical screening platforms to ensure that only the most efficacious drug candidates move on to in vivo testing.

The majority of drugs are typically orally administered. The journey from drug discovery to approval is often long and expensive, involving multiple stages. A major challenge in the drug development process is drug-induced liver injury (DILI), a condition that affects the liver, the organ responsible for metabolizing most drugs. Traditionally, identifying DILI risk has been difficult due to the poor correlation between preclinical animal models and in vitro systems. Differences in physiology between humans and animals or cell lines contribute to the failure of many drug programs during clinical trials. The use of advanced in vitro systems that closely mimic human physiology, such as organ-on-a-chip models like gut-liver-on-a-chip, can be crucial in improving drug efficacy while minimizing toxicity. Additionally, the adaptation of these technologies has the potential to significantly reduce both the time and cost associated with obtaining safe drug approvals, all while adhering to the 3Rs principle (replacement, reduction, refinement). In this review, we discuss the significance, current status, and future prospects of advanced platforms, specifically organ-on-a-chip models, in supporting preclinical drug discovery.

Three-dimensional (3D) tissue culture models provide in vivo-like conditions for studying cell physiology. This study aimed to examine the efficiency of pyramidal microwell geometries in microfluidic devices on spheroid formation, cell growth, viability, and differentiation in mouse embryonic stem cells (mESCs). The static culture using the hanging drop (HD) method served as a control. The microfluidic chips were fabricated to have varying pyramidal tip angles, including 66°, 90°, and 106°. From flow simulations, when the tip angle increased, streamline distortion decreased, resulting in more uniform flow and a lower velocity gradient near the spheroids. These findings demonstrate the significant influence of microwell geometry on fluid dynamics. The 90° microwells provide optimal conditions, including uniform flow and reduced shear stress, while maintaining the ability for waste removal, resulting in superior spheroid growth compared to the HD method and other microwell designs. From the experiments, by Day 3, spheroids in the 90° microwells reached approximately 400 µm in diameter which was significantly larger than those in the 66° microwells, 106° microwells, and HD cultures. Brachyury gene expression in the 90° microwells was four times higher than the HD method, indicating enhanced mesodermal differentiation essential for cardiac differentiation. Immunofluorescence staining confirmed cardiomyocyte differentiation. In conclusion, microwell geometry significantly influences 3D cell culture outcomes. The pyramidal microwells with a 90° tip angle proved most effective in promoting spheroid growth and cardiac differentiation of mESC differentiation, providing insights for optimizing microfluidic systems in tissue engineering and regenerative medicine.

Dimethyl sulfoxide (DMSO) is widely used as an adjuvant in dissolving insoluble compounds in an aqueous medium; however, it can induce significant molecular changes in cells. The possible damages may occur obeying a tissue-specific profile, and the effect on human apical papilla cells (hAPC) remains unknown. Therefore, this study aimed to evaluate DMSO effects on the viability and mineralization activity in hAPC cultures in vitro and to establish standards of maximum concentrations for its use in laboratory routines. hAPCs were cultured, plated, and maintained in media containing increasing concentrations of Dimethyl sulfoxide (0.1%, 0.5%, 1%, 5%, and 10%) for 24 h, 48 h, 72 h, and 7 days. At each time point, the cells were subjected to the MTT assay. The Alizarin red S staining assay was performed to evaluate the osteo/odontogenic mineralization potential of hAPC DMSO-exposed (0.1%, 0.5%, and 1%) in the 21-day time-point. Statistical analysis was performed using one-way analysis of variance followed by Tukey's post hoc test (p<0.05). In general, the 5% and 10% DMSO concentrations were shown to be cytotoxic for hAPC at all analyzed time points, and the hAPC DMSO-stimulated presented higher osteo/odontogenic mineralization potential. Therefore, the 5% and 10% DMSO concentrations should be avoided, and the mineralization activity assay should be carefully designed in order to avoid biases at in vitro assays using hAPC cultures.

Melanoma malignum is considered the most dangerous form of skin cancer, characterized by the exceptional resistance to many conventional chemotherapies. The aim of this study was to evaluate the effect of NutramilTM Complex (NC)-Food for Special Medical Purpose (FSMP), on two types of melanoma cell lines, primary WM115 and malignant WM266-4.

At 24 h after seeding, growth medium was replaced with a medium containing encoded treatments of NC or NC-CC (NutramilTM Complex without calcium caseinate) at various concentrations. Cells were treated for 24, 48, and 72 h.

Our results showed that NutramilTM Complex reduces proliferation of malignant melanoma WM266-4 cells but did not affect the proliferation of WM115 primary melanoma. This was followed by measured down-regulation of selected pro-survival proteins expression in WM266-4 cells, specifically ERK1/2, AKT-1, HSP27, Survivin, and TAK1. Interestingly, our results showed elevated levels of some pro-apoptotic proteins in both cell lines, including Bad, Smad2, p38MAPK, cleaved forms of Caspase-3/7, as well as cleaved PARP.

Taken together, our results indicate that various melanoma cancer cell lines may respond in a different way to the same compound. They also suggest induction of apoptotic pathway by NutramilTM Complex as the most likely mechanism of its anticarcinogenic activity.

Microtissues, engineered to emulate the complexity of human organs, are revolutionizing the fields of regenerative medicine, disease modelling, and drug screening. Despite the promise of traditional microtissue engineering, it has yet to achieve the precision required to fully replicate organ-like structures. Enter 3D bioprinting, a transformative approach that offers unparalleled control over the microtissue's spatial arrangement and mechanical properties. This cutting-edge technology enables the detailed layering of bioinks, crafting microtissues with tissue-like 3D structures. It allows for the direct construction of organoids and the fine-tuning of the mechanical forces vital for tissue maturation. Moreover, 3D-printed devices provide microtissues with the necessary guidance and microenvironments, facilitating sophisticated tissue interactions. The applications of 3D-printed microtissues are expanding rapidly, with successful demonstrations of their functionality in vitro and in vivo. This technology excels at replicating the intricate processes of tissue development, offering a more ethical and controlled alternative to traditional animal models. By simulating in vivo conditions, 3D-printed microtissues are emerging as powerful tools for personalized drug screening, offering new avenues for pharmaceutical development and precision medicine.

Bone and cartilage tissues are essential for movement and structure, yet diseases like osteoarthritis affect millions. Traditional therapies have limitations, necessitating innovative approaches. Organoid technology, leveraging stem cells' regenerative potential, offers a novel platform for disease modelling and therapy. This review focuses on advancements in bone/cartilage organoid technology, highlighting the role of stem cells, biomaterials, and external factors in organoid development. We discuss the implications of these organoids for regenerative medicine, disease research, and personalised treatment strategies, presenting organoids as a promising avenue for enhancing cartilage repair and bone regeneration. Bone/cartilage organoids will play a greater role in the treatment of bone/cartilage diseases in the future, and promote the progress of biological tissue engineering.

Metabolic dysfunction-associated fatty liver disease (MASLD) presents a growing global health challenge with limited therapeutic choices. This review delves into the array of ex vivo tools and models utilized in MASLD research, encompassing liver-on-a-chip (LoC) systems, organoid-derived tissue-like structures, and human precision-cut liver slice (PCLS) systems. Given the urgent need to comprehend MASLD pathophysiology and identify novel therapeutic targets, this paper aims to shed light on the pivotal role of advanced ex vivo models in enhancing disease understanding and facilitating the development of potential therapies. Despite challenges posed by the elusive disease mechanism, these innovative methodologies offer promise in reducing the utilization of in vivo models for MASLD research while accelerating drug discovery and biomarker identification, thereby addressing critical unmet clinical needs.

Targeted cancer therapy (TCT) is gaining increased interest because it reduces the risks of adverse side effects by specifically treating tumor cells. TCT testing has traditionally been performed using two-dimensional (2D) cell culture and animal studies. Organ-on-a-chip (OoC) platforms have been developed to recapitulate cancer in vitro, as cancer-on-a-chip (CoC), and used for chemotherapeutics development and testing. This review explores the use of CoCs to both develop and test TCTs, with a focus on three main aspects, the use of CoCs to identify target biomarkers for TCT development, the use of CoCs to test free, un-encapsulated TCTs, and the use of CoCs to test encapsulated TCTs. Despite current challenges such as system scaling, and testing externally triggered TCTs, TCToC shows a promising future to serve as a supportive, pre-clinical platform to expedite TCT development and bench-to-bedside translation.

Recently approved RET tyrosine kinase inhibitors (TKIs) have shown promising therapeutic effects against RET-rearranged non-small cell lung cancer (NSCLC) or RET-mutated thyroid cancer. However, resistance develops, limiting long-term efficacy. Although many RET-TKI resistance mechanisms, such as secondary mutations in RET or activation of bypass pathways, are known, some primary or acquired resistance mechanisms are unclear. Here, human genome-wide CRISPR/Cas9 screening was performed to identify genes related to drug-tolerant persister cells. Patient-derived cells with RET-fusion were introduced genome-wide sgRNA library and treated with RET-TKI for 9 days, resulting in the discovery of several candidate genes. Knockout of MED12 or MIG6 significantly increased residual drug-tolerant persister cells under RET-TKI treatment. MIG6 loss induced significant EGFR activation even with low concentrations of EGFR ligands and led to resistance to RET-TKIs. EGFR inhibition with afatinib or cetuximab in combination with RET TKIs was effective in addressing drug persistence. By contrast, a KIF5B-RET positive cells established from a RET-rearranged NSCLC patient, showed significant resistance to RET-TKIs and high dependence on EGFR bypass signaling. Consistently, knocking out EGFR or RET led to high sensitivity to RET or EGFR inhibitor respectively. Here, we have provided a comprehensive analysis of adaptive and acquired resistance against RET-rearranged NSCLC.

Endothelial cells are sensitive to mechanical force and can convert it into biochemical signals to trigger mechano-chemo-transduction. Although conventional techniques have been used to investigate the subsequent modifications of cellular expression after mechanical stimulation, the in situ and real-time acquiring the transient biochemical information during mechanotransduction process remains an enormous challenge. In this work, we develop a flexible and multi-functional three-dimensional conductive scaffold that integrates cell growth, mechanical stimulation, and electrochemical sensing by in situ growth of enokitake-like Au nanowires on a three-dimensional porous polydimethylsiloxane substrate. The conductive scaffold possesses stable and desirable electrochemical sensing performance toward nitric oxide under mechanical deformation. The prepared e-AuNWs/CC/PDMS scaffold exhibits a good electrocatalytic ability to NO with a linear range from 2.5 nM to 13.95 μM and a detection limit of 8 nM. Owing to the excellent cellular compatibility, endothelial cells can be cultured directly on the scaffold and the real-time inducing and recording of nitric oxide secretion under physiological and pathological conditions were achieved. This work renders a reliable sensing platform for real-time monitoring cytomechanical signaling during endothelial mechanotransduction and is expected to promote other related biological investigations based on three-dimensional cell culture.

This review centers on the synthesis and characterization of both natural and synthetic hydrogels, highlighting their diverse applications across various fields. We will delve into the evolution of hydrogels, focusing on the importance of polysaccharide-based and synthetic variants, which have been particularly chosen for 3D spheroid development in cancer research and drug delivery. A detailed background on the research and specific methodologies, including the in-situ free radical polymerization used for synthesizing these hydrogels, will be extensively discussed. Additionally, the review will explore various applications of these hydrogels, such as their self-healing properties, swelling ratios, pH responsiveness, and cell viability. A comprehensive literature review will support this investigation. Ultimately, this review aims to clearly outline the objectives and significance of hydrogel synthesis and their applications.

Cardiovascular diseases (CVDs) are leading causes of morbidity and mortality globally, characterized by complications such as heart failure, atherosclerosis, and coronary artery disease. The vascular endothelium, forming the inner lining of blood vessels, plays a pivotal role in maintaining vascular homeostasis. The dysfunction of endothelial cells contributes significantly to the progression of CVDs, particularly through impaired cellular communication and paracrine signaling with other cell types, such as smooth muscle cells and macrophages. In recent years, co-culture systems have emerged as advanced in vitro models for investigating these interactions and mimicking the pathological environment of CVDs. This review provides an in-depth analysis of co-culture models that explore endothelial cell dysfunction and the role of cellular interactions in the development of vascular diseases. It summarizes recent advancements in multicellular co-culture models, their physiological and therapeutic relevance, and the insights they provide into the molecular mechanisms underlying CVDs. Additionally, we evaluate the advantages and limitations of these models, offering perspectives on how they can be utilized for the development of novel therapeutic strategies and drug testing in cardiovascular research.

Long-term culture of primary lymphocytes and hematopoietic stem and progenitor cells (HSPCs) is pivotal to their expansion and study. Furthermore, genetic engineering of the above-mentioned primary human cells has several safety needs, including the requirement of efficient in vitro assays for unwanted tumorigenic events. In this work, we tested and optimized the Miniaturized Optically Accessible Bioreactor (MOAB) platform. The MOAB consists of a millifluidic cell culture device with three optically-accessible culture chambers. Inside the MOAB, we inserted a silk-based framework that resembles some properties of the bone marrow environment and cultivated in this device either CD4+ T lymphocytes isolated from healthy donor buffy coat or cord blood-derived hematopoietic CD34+ cells. A fraction of these cells is viable for up to 3 months. Next, we tested the capability of the MOAB to detect tumorigenic events. Serial dilutions of engineered fluorescent tumor cells were mixed with either CD4+ or CD34+ primary cells, and their growth was followed. By this approach, we successfully detected as little as 100 tumorigenic cells mixed with 100,000 primary cells. We found that non-tumorigenic primary cells colonized the silk environment, whereas tumor cells, after an adaptation phase, expanded and entered the circulation. We conclude that the millifluidic platform allows the detection of rare tumorigenic events in the long-term culture of human cells.

As one of the leading causes of global mortality and morbidity, various neurological diseases cause social and economic burdens. Despite significant advances in the treatment of neurological diseases, establishing a proper disease model, especially for degenerative and infectious diseases, remains a major challenging issue. For long, mice were the model of choice but suffered from serious drawbacks of differences in anatomical and functional aspects of the nervous system. Furthermore, the collection of postmortem brain tissues limits their usage in cultured cell lines. Overcoming such limitations has prompted the usage of stem cells derived from the peripheral nervous system, such as the cells of the olfactory mucosa as a preferred choice. These cells can be easily cultured in vitro and retain the receptors of neuronal cells life-long. Such cells have various advantages over embryonic or induced stem cells, including homology, and ease of culture and can be conveniently obtained from diseased individuals through either biopsies or exfoliation. They have continuously helped in understanding the genetic and developmental mechanisms of degenerative diseases like Alzheimer's and Parkinson's disease. Moreover, the mode of infection of various viruses that can lead to postviral olfactory dysfunction, such as the Zika virus can be monitored through these cells in vitro and their therapeutic development can be fastened.

Several organ-on-chip and cell-on-chip devices have been reported, however, their main drawback is that they are not interoperable (i.e., they have been fabricated with customized equipment, thus cannot be applied in other facilities, unless having the same setup), and require cell-culture facilities and benchtop instrumentation. As a consequence, results obtained with such devices do not generally comply with the principles of findability, accessibility, interoperability, and reusability (FAIR). To overcome such limitation, leveraging cost-effective 3D printing we developed a bioluminescent tissue on-a-chip device that can be easily implemented in any laboratory. The device enables continuous monitoring of cell co-cultures expressing different bioluminescent reporter proteins and, thanks to the implementation of new highly bioluminescent luciferases having high pH and thermal stability, can be monitored via smartphone camera. Another relevant feature is the possibility to insert the chip into a commercial 24-well plate for use with standard benchtop instrumentation. The suitability of this device for 3D cell-based biosensing for monitoring activation of target molecular pathways, i.e., the inflammatory pathway via nuclear factor kappa-B (NF-κB) activation, and general cytotoxicity is here reported showing similar analytical performance when compared to conventional 3D cell-based assays performed in 24-well plates.

The dysregulation of pH has been linked to the onset of chronic conditions, such as cancer and neurological diseases. Consequently, the development of a highly sensitive tool for intracellular pH sensing is imperative to investigate the interplay between pH and the biochemical changes accompanying disease pathogenesis. Here, we present the development of a ratiometric fluorescent nanoprobe, NpRhoDot, designed for precisely measuring pH levels. We demonstrate its efficacy in sensitively reporting intracellular pH in monolayer A549 lung cancer cells, primary fibroblast cells, and 3D tumor spheroids derived from the DLD-1 colorectal adenocarcinoma cell line. NpRhoDot leverages a novel design, where stable carbon dots are functionalized with a pH-responsive ratiometric fluorescent probe comprising a naphthalimide-rhodamine moiety, NpRho1. This design confers NpRhoDot with the high pH sensitivity characteristics of organic fluorescent probes, along with excellent photostability up to 1 h and biocompatibility of carbon dots. Through one-photon and two-photon fluorescence microscopy, we validate the reliability of NpRhoDot for biosensing intracellular pH in monolayer and three-dimensional tumor models from pH 4 to 7.

Cancer is a collection of illnesses characterized by aberrant cellular proliferation that can infiltrate or metastasize to distant anatomical sites, posing a notable threat to human well-being due to its substantial morbidity and death rates worldwide. The potential of plant-derived natural compounds as anticancer medicines has been assessed owing to their favorable attributes of few side effects and significant antitumor activity. Mangrove plants and their derived compounds have been scientifically shown to exhibit many significant beneficial biological activities, such as anti-inflammatory, immunomodulatory, antioxidant, neuroprotective, cardioprotective, and hepatoprotective properties. This study summarized mangrove plants and their derived compounds as potential anticancer agents, with an emphasis on the underlying molecular mechanisms. To explore this, we gathered data on the preclinical (in vivo and in vitro) anticancer effects of mangrove plants and their derived compounds from reputable literature spanning 2000 to 2023. We conducted thorough searches in various academic databases, including PubMed, ScienceDirect, Wiley Online, SpringerLink, Google Scholar, Scopus, and the Web of Science. The results demonstrated that mangrove plants and their derived compounds have promising anticancer properties in preclinical pharmacological test systems through various molecular mechanisms, including induction of oxidative stress and mitochondrial dysfunction, cytotoxicity, genotoxicity, cell cycle arrest, apoptosis, autophagy, antiproliferative, antimetastatic, and other miscellaneous actions. Upon thorough observation of the pertinent information, it is suggested that mangrove plants and their derived chemicals may serve as a potential lead in the development of novel drugs for cancer therapy.

Bladder cancer (BCa) is one of the most lethal genitourinary malignancies owing to its propensity for recurrence and poor survival. The biochemical pathway, signal transducer and activator of transcription 3 (STAT3), has gained significance as a molecular pathway that promotes proliferation, invasion, and chemoresistance. In this study, we explored the targeting of STAT3 with TTI-101 and SH5-07 in BCa and elucidated the mechanisms in three-dimensional (3D) spheroid and organoid models. We optimized the growth of spheroids from human, rat, and mouse BCa cell lines (J82, NBT-II, and MB49 respectively) and organoids from BBN (N-butyl-N-(4-hydroxybutyl)-nitrosamine)-induced rat bladder tumors. Cell viability was assessed using MTT and trypan blue assays. Intracellular ATP production, ROS production, and calcium AM (CA)/EtBr staining were used to measure the spheroid and organoid inhibition and mitochondrial function. Western blot analysis was performed to evaluate the pharmacodynamic markers involved in cell proliferation, apoptosis, cancer stem cells (CSCs), and STAT3 signaling in BCa. We found that targeting STAT3 (using TTI-101 and SH5-07) significantly reduced the proliferation of BCa spheroids and organoids, which was accompanied by decreased expression of pSTAT3, Cyclin D1, and PCNA. Our data also demonstrated that treatment with STAT3 inhibitors induced ROS production and cell death in BCa spheroids and organoids. STAT3 inhibition-induced cell death was associated with the activation of caspase 3/7 and PARP cleavage. Moreover, TTI-101 and SH5-07 target cancer stem cells by downregulating the expression of CD44 and CD133 in 3D models. This study provides the first evidence for the prevention of BCa with small-molecule inhibitors TTI-101 and SH5-07 via suppression of CSCs and STAT3 signaling.

The rotating-wall vessel (RWV) bioreactor, a 3D suspension culture system, faces challenges related to non-uniform tissue growth during the incubation of bone and heart tissues. Okra mucilage, an extract from okra pods with non-Newtonian rheological properties, has shown potential as a plasma replacement agent and has no induced cytotoxic effects. In this study, we investigated the flow structure of okra mucilage in rotating wall vessel system. By modifying the RWV and adding okra mucilage, we analyzed the flow structure using a high-speed camera and particle image velocimetry (PIV). Our results showed that okra mucilage creates a concentric circle-shaped rigid-like rotation at all rotation speeds (1-50 rpm). The high viscosity of okra mucilage resulted in a low terminal velocity for microparticles and quick response to rotational movements. These findings suggest that okra mucilage has the potential to enhance the uniformity of tissue growth in RWV systems by stabilizing the flow structure and reducing microparticle sedimentation.

Pancreatic ductal adenocarcinoma (PDAC) is the most common type of pancreatic cancer, a leading cause of cancer-related deaths globally. Initial lesions of PDAC develop within the exocrine pancreas' functional units, with tumor progression driven by interactions between PDAC and stromal cells. Effective therapies require anatomically and functionally relevantin vitrohuman models of the pancreatic cancer microenvironment. We employed tomographic volumetric bioprinting, a novel biofabrication method, to create human fibroblast-laden constructs mimicking the tubuloacinar structures of the exocrine pancreas. Human pancreatic ductal epithelial (HPDE) cells overexpressing the KRAS oncogene (HPDE-KRAS) were seeded in the multiacinar cavity to replicate pathological tissue. HPDE cell growth and organization within the structure were assessed, demonstrating the formation of a thin epithelium covering the acini inner surfaces. Immunofluorescence assays showed significantly higher alpha smooth muscle actin (α-SMA) vs. F-actin expression in fibroblasts co-cultured with cancerous versus wild-type HPDE cells. Additionally,α-SMA expression increased over time and was higher in fibroblasts closer to HPDE cells. Elevated interleukin (IL)-6 levels were quantified in supernatants from co-cultures of stromal and HPDE-KRAS cells. These findings align with inflamed tumor-associated myofibroblast behavior, serving as relevant biomarkers to monitor early disease progression and target drug efficacy. To our knowledge, this is the first demonstration of a 3D bioprinted model of exocrine pancreas that recapitulates its true 3-dimensional microanatomy and shows tumor triggered inflammation.

Responding to global standards and legislative updates in Canada, including Bill S-5 (2023), toxicity testing is shifting towards more ethical, in vitro methods. Traditional two-dimensional (2D) monolayer cell cultures, limited in replicating the complex in vivo environment, have prompted the development of more relevant three-dimensional (3D) spheroidal hepatocyte cultures. This study introduces the first 3D spheroid model for McA-RH7777 cells, assessing xenobiotic receptor activation, cellular signaling, and toxicity against dexamethasone and naphthenic acid (NA)-fraction components; NAFCs. Our findings reveal that 3D McA-RH7777 spheroids demonstrate enhanced sensitivity and more uniform dose-response patterns in gene expression related to xenobiotic metabolism (AhR and PPAR) for both single compounds and complex mixtures. Specifically, 3D cultures showed significant gene expression changes upon dexamethasone exposure and exhibited varying degrees of sensitivity and resistance to the apoptotic effects induced by NAFCs, in comparison to 2D cultures. The optimization of 3D culture conditions enhances the model's physiological relevance and enables the identification of genomic signatures under varied exposures. This study highlights the potential of 3D spheroid cultures in providing a more accurate representation of the liver's microenvironment and advancing our understanding of cellular mechanisms in toxicity testing.

The high incidence of malignant melanoma highlights the need forin vitromodels that accurately represent the tumour microenvironment, enabling developments in melanoma therapy and drug screening. Despite several advancements in 3D cell culture models, appropriate melanoma models for evaluating drug efficacy are still in high demand. The 3D pneumatic extrusion-based bioprinting technology offers numerous benefits, including the ability to achieve high-throughput capabilities. However, there is a lack of research that combines pneumatic extrusion-based bioprinting with analytical assays to enable efficient drug screening in 3D melanoma models. To address this gap, this study developed a simple and highly reproducible approach to fabricate a 3D A375 melanoma cell culture model using the pneumatic extrusion-based bioprinting technology. To optimise this method, the bioprinting parameters for producing 3D cell cultures in a 96-well plate were adjusted to improve reproducibility while maintaining the desired droplet size and a cell viability of 92.13 ± 6.02%. The cross-linking method was optimised by evaluating cell viability and proliferation of the 3D bioprinted cells in three different concentrations of calcium chloride. The lower concentration of 50 mM resulted in higher cell viability and increased cell proliferation after 9 d of incubation. The A375 cells exhibited a steadier proliferation rate in the 3D bioprinted cell cultures, and tended to aggregate into spheroids, whereas the 2D cell cultures generally formed monolayered cell sheets. In addition, we evaluated the drug responses of four different anti-cancer drugs on the A375 cells in both the 2D and 3D cell cultures. The 3D cell cultures exhibited higher levels of drug resistance in all four tested anti-cancer drugs. This method presents a simple and cost-effective method of producing and analysing 3D cell culture models that do not add additional complexity to current assays and shows considerable potential for advancing 3D cell culture models' drug efficacy evaluations.

Rheumatoid arthritis (RA) is a complex condition that is influenced by various causes, including immunological, genetic, and environmental factors. Several studies using animal models have documented immune system dysfunction and described the clinical characteristics of the disease. These studies have provided valuable insights into the pathogenesis of inflammatory arthritis and the identification of new targets for treatment. Nevertheless, none of these animal models successfully replicated all the characteristics of RA. Additionally, numerous experimental medications, which were developed based on our enhanced comprehension of the immune system's function in RA, have shown potential in animal research but ultimately proved ineffective during different stages of clinical trials. There have been several novel therapy alternatives, which do not achieve a consistently outstanding therapeutic outcome in all patients. This underscores the importance of employing the progress in in vitro models, particularly 3D models like tissue explants, and diverse multicomponent approaches such as coculture strategies, synovial membrane, articular cartilage, and subchondral bone models that accurately replicate the structural characteristics of RA pathophysiology. These methods are crucial for the advancement of potential therapeutic strategies. This review discusses the latest advancements in in vitro models and their potential to greatly impact research on managing RA.

Intramuscular (IM) injections deliver a plethora of drugs. The majority of IM-related literature details dissolution and/or pharmacokinetic (PK) studies, using methods with limited assessments of post-injection events that can impact drug fate, and absorption parameters. Food and Drug Association guidelines no longer require preclinical in vivo modeling in the U.S.A. Preclinical animal models fail to correlate with clinical outcomes, highlighting the need to study, and understand, IM drug fate in vitro using bespoke models emulating human IM sites. Post-IM injection events, i.e. underlying processes that influence PK outcomes, remain unacknowledged, complicating the application of in vitro methods in preclinical drug development. Understanding such events could guide approaches to predict and modulate IM drug fate in humans.

This article reviews challenges in biorelevant IM site modeling (i.e. modeling drug fate outcomes), the value of technologies available for developing IM injectables, methods for studying drug fate, and technologies for training in performing IM administrations. PubMed, Web-of-Science, and Lens databases provided papers published between 2014 and 2024.

IM drug research is expanding what injectable therapeutics can achieve. However, post-injection events that influence PK outcomes remain poorly understood. Until addressed, advances in IM drug development will not realize their full potential.

A mechanism exploration is an important part of toxicological studies. However, traditional cell and animal models can no longer meet the current needs for in-depth studies of toxicological mechanisms. The three-dimensional (3D) organoid derived from human embryonic stem cells (hESC) or induced pluripotent stem cells (hiPSC) is an ideal experimental model for the study of toxicological effects and mechanisms, which further recapitulates the human tissue microenvironment and provides a reliable method for studying complex cell-cell interactions. This article provides a comprehensive overview of the state of the 3D organoid technology in toxicological studies, including a bibliometric analysis of the existing literature and an exploration of the latest advances in toxicological mechanisms. The use of 3D organoids in toxicology research is growing rapidly, with applications in disease modeling, organ-on-chips, and drug toxicity screening being emphasized, but academic communications among countries/regions, institutions, and research scholars need to be further strengthened. Attempts to study the toxicological mechanisms of exogenous chemicals such as heavy metals, nanoparticles, drugs and organic pollutants are also increasing. It can be expected that 3D organoids can be better applied to the safety evaluation of exogenous chemicals by establishing a standardized methodology.

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.

The aim of this study was to develop a three-dimensional (3D) cell model in order to evaluate the effectiveness of a traditional Chinese medicine decoction in the treatment of arthritis. Chondrocytes (ATDC5) and osteoblasts (MC3T3-E1) were 3D printed separately using methacryloyl gelatin (GelMA) hydrogel bioinks to mimic the natural 3D cell environment. Both cell types showed good biocompatibility in GelMA. Lipopolysaccharide (LPS) was added to the cell models to create inflammation models, which resulted in increased expression of inflammatory factors IL-1β, TNF-α, iNOS, and IL-6, and decreased expression of cell functional genes such as Collagen II (COLII), transcription factor SOX-9 (Sox9), Aggrecan, alkaline phosphatase (ALP), RUNX family transcription factor 2 (Runx2), Collagen I (COLI), Osteopontin (OPN), and bone morphogenetic protein-2 (BMP-2). The created inflammation model was then used to evaluate the effectiveness of Dangguiniantongtang (DGNT) decoctions. The results showed that DGNT reduced the expression of inflammatory factors and increased the expression of functional genes in the cell model. In summary, this study established a 3D cell model to assess the effectiveness of traditional Chinese medicine (TCM) decoctions, characterized the gene expression profile of the inflammatory state model, and provided a practical reference for future research on TCM efficacy evaluation for arthritis treatment.

The SARS-CoV-2 pandemic has spurred an unparalleled scientific endeavor to elucidate the virus' structure, infection mechanisms, and pathogenesis. Two-dimensional culture systems have been instrumental in shedding light on numerous aspects of COVID-19. However, these in vitro systems lack the physiological complexity to comprehend the infection process and explore treatment options. Three-dimensional (3D) models have been proposed to fill the gap between 2D cultures and in vivo studies. Specifically, spheroids, composed of lung cell types, have been suggested for studying SARS-CoV-2 infection and serving as a drug screening platform.

3D lung spheroids were prepared by coculturing human alveolar or bronchial epithelial cells with human lung stromal cells. The morphology, size, and ultrastructure of spheroids before and after SARS-CoV-2 infection were analyzed using optical and electron microscopy. Immunohistochemistry was used to detect spike protein and, thus, the virus presence in the spheroids. Multiplex analysis elucidated the cytokine release after virus infection.

The spheroids were stable and kept their size and morphology after SARS-CoV-2 infection despite the presence of multivesicular bodies, endoplasmic reticulum rearrangement, tubular compartment-enclosed vesicles, and the accumulation of viral particles. The spheroid responded to the infection releasing IL-6 and IL-8 cytokines.

This study demonstrates that coculture spheroids of epithelial and stromal cells can serve as a cost-effective infection model for the SARS-CoV-2 virus. We suggest using this 3D spheroid as a drug screening platform to explore new treatments related to the cytokines released during virus infection, especially for long COVID treatment.