The progressive loss of dopaminergic neurons in affected patient brains is one of the pathological features of Parkinson's disease, the second most common human neurodegenerative disease. Although the detailed pathogenesis accounting for dopaminergic neuron degeneration in Parkinson's disease is still unclear, the advancement of stem cell approaches has shown promise for Parkinson's disease research and therapy. The induced pluripotent stem cells have been commonly used to generate dopaminergic neurons, which has provided valuable insights to improve our understanding of Parkinson's disease pathogenesis and contributed to anti-Parkinson's disease therapies. The current review discusses the practical approaches and potential applications of induced pluripotent stem cell techniques for generating and differentiating dopaminergic neurons from induced pluripotent stem cells. The benefits of induced pluripotent stem cell-based research are highlighted. Various dopaminergic neuron differentiation protocols from induced pluripotent stem cells are compared. The emerging three-dimension-based brain organoid models compared with conventional two-dimensional cell culture are evaluated. Finally, limitations, challenges, and future directions of induced pluripotent stem cell-based approaches are analyzed and proposed, which will be significant to the future application of induced pluripotent stem cell-related techniques for Parkinson's disease.

Surface topography or curvature plays a crucial role in regulating cell behavior, influencing processes such as adhesion, proliferation, and gene expression. Recent advancements in nano- and micro-fabrication techniques have enabled the development of biomimetic systems that mimic native extracellular matrix (ECM) structures, providing new insights into cell-adhesion mechanisms, mechanotransduction, and cell-environment interactions. This review examines the diverse applications of engineered topographies across multiple domains, including antibacterial surfaces, immunomodulatory devices, tissue engineering scaffolds, and cancer therapies. It highlights how nanoscale features like nanopillars and nanospikes exhibit bactericidal properties, while many microscale patterns can direct stem cell differentiation and modulate immune cell responses. Furthermore, we discuss the interdisciplinary use of topography for combined applications, such as the simultaneous regulation of immune and tissue cells in 2D and 3D environments. Despite significant advances, key knowledge gaps remain, particularly regarding the effects of topographical cues on multicellular interactions and dynamic 3D contexts. This review summarizes current fabrication methods, explores specific and interdisciplinary applications, and proposes future research directions to enhance the design and utility of topographically patterned biomaterials in clinical and experimental settings.

Microfluidic chip-based electrochemical sensors have been developed to detect cancer biomarkers and monitor changes in the tumor microenvironment. However, the limitation of detecting only a single biomarker restricts their utility as accurate diagnostic tools. Simultaneous detection of multiple tumor biomarkers is important for early diagnosis of cancer. In this work, we report the development of a microfluidic-based electrochemical immunosensor platform capable of simultaneously observing multiple biomarkers expressed by three dimensions (3D) cell spheroids. The sensor platform employs a nanocomposite electrode material consisting of gold nanoparticles and carbon nanotubes, which enables sensitive and selective detection. The sensor was fabricated using 3D and printed circuit boards (PCB) printing techniques, demonstrating the feasibility of cost-effective manufacturing. The developed platform was able to quantitatively detect two key cancer biomarkers, carcinoembryonic antigen (CEA) and Ki67, with limits of detection of 0.97 ng/mL for each. Furthermore, the sensor was successfully utilized to observe the knockdown of these biomarkers, showcasing its potential for both diagnostic and therapeutic monitoring applications. These results suggest that the presented electrochemical sensor platform provides a promising lab-on-a-chip technology for comprehensive 3D cell spheroid-based cancer biomarker analysis, which could have significant implications for future clinical diagnostics and personalized medicine.

Corneal stromal defects represent a significant global cause of blindness, necessitating innovative therapeutic strategies to address the limitations of conventional treatments, such as corneal transplantation. Tissue engineering, a cornerstone of regenerative medicine, offers a transformative approach by leveraging biomaterial-based solutions to restore damaged tissues. Among these, three-dimensional (3D) scaffolds fabricated using advanced techniques like 3D printing have emerged as a promising platform for corneal regeneration. These scaffolds replicate the native extracellular matrix (ECM) architecture, providing a biomimetic microenvironment that supports cell proliferation, differentiation, and tissue integration. This review highlights recent advances in the design and fabrication of 3D scaffolds for corneal stroma engineering (CSE), emphasizing the critical interplay between scaffold architecture, mechanical properties, and bioactive signaling in directing cellular behavior and tissue regeneration. Likewise, we emphasize the diverse range of biomaterials utilized in scaffold fabrication, highlighting their influence on cellular interactions and tissue reconstruction. By elucidating the complex relationship between scaffold design and biologics, this review aims to illuminate the evolution of next-generation strategies for engineering functional corneal tissue. Eventually, this review will provide a comprehensive synthesis of the current state-of-the-art in 3D scaffold-based corneal tissue engineering (CTE), offering insights that could advance progress toward effective vision restoration therapies.

Peripheral nervous system (PNS) regeneration is a rapidly advancing field with critical implications for addressing sensory impairments and neuropathic conditions. Dorsal root ganglion (DRG) neurons, essential for sensory transmission, exhibit regenerative potential through axonal regeneration. However, the mechanisms driving these processes are not yet understood. This study introduces an innovative 3D-bioprinted fibroblasts/DRG co-culture construct, specifically designed to investigate and characterize PNS regeneration and wiring mechanisms under both physiological and pathophysiological conditions. By characterizing bioink rheology and optimizing bioprinting parameters, we created a stable, biocompatible derma-like construct supporting cell adhesion and growth. Bioprinted 3T3 fibroblasts demonstrate high viability and proliferation, while DRG neurons exhibit enhanced neurite outgrowth and complex branching patterns within the co-culture system. These findings highlight the role of fibroblasts in promoting axonal regeneration and provide a robust in vitro platform for studying sensory system reinnervation. This model lays the foundation for developing personalized therapies for neuropathic pain and sensory dysfunction, advancing both fundamental neuroscience and translational medicine.

The industrialization of mesenchymal stem cells for regenerative medicine faces substantial challenges, particularly in large-scale production. Conventional 2-dimensional (2D) culture systems demonstrate limitations in meeting clinical requirements, such as inadequate cell yield, and poor cell-cell and cell-matrix interactions. These challenges can potentially be addressed by employing a 3D culture platform, which offers higher cell yields and enhanced efficacy. Moreover, it is essential to conduct a systematic and rigorous evaluation of cells produced in 3D culture systems to ensure their successful clinical translation. In this study, we cultured human umbilical cord mesenchymal stem cells (hUCMSCs) using an automated, scalable, and enclosed 3D microcarrier-bioreactor system, and comprehensively investigated their biological characteristics and potential therapeutic effects for diabetic wound repair. Our findings revealed that hUCMSCs harvested from this 3D microcarrier-bioreactor system are genetically stable and maintain the trilineage differentiation potential. Compared to hUCMSCs expanded under 2D conditions, those cultured in 3D exhibited reduced senescence and enhanced capabilities in migration, angiogenesis, and anti-inflammatory responses across different passages in vitro. RNA-sequencing analysis showed higher expression levels of genes related to angiogenesis and anti-inflammatory pathways in hUCMSCs cultured in 3D compared to those in 2D, which was further validated using quantitative real-time polymerase chain reaction and Western blot analysis. Additionally, 3D-cultured hUCMSCs demonstrated superior therapeutic effects for diabetic wound repair in mice, potentially due to their enhanced angiogenetic and anti-inflammatory effects. Collectively, our finding showcases the high quality of hUCMSCs cultured using an automated and enclosed 3D microcarrier-bioreactor system and their promising potential for diabetic wound repair.

Interfaces between distinct tissues or between tissues and environments are common in multicellular organisms. The evolution and stability of these interfaces are essential for tissue development, and their dysfunction can lead to diseases such as cancer. Mounting efforts, either theoretical or experimental, have been devoted to uncovering the morphodynamics of tissue interfaces. Here, we review the recent progress of studies on interface morphodynamics. The regulatory mechanisms governing interface evolution are dissected, with a focus on adhesion, cortical tension, cell activity, extracellular matrix, and microenvironment. We examine the methodologies used to study morphodynamics, emphasizing the characteristics of experimental techniques and theoretical models. Finally, we explore the broader implications of interface morphodynamics in tissue morphogenesis and diseases, offering a comprehensive perspective on this rapidly developing field.

Liver fibrosis is a reversible but complex pathological condition associated with chronic liver diseases, affecting over 1.5 billion people worldwide. It is characterized by excessive extracellular matrix deposition resulting from sustained liver injury, often advancing to cirrhosis and cancer. As its progression involves various cell types and pathogenic factors, understanding the intricate mechanisms is essential for the development of effective therapies. In this context, extensive efforts have been made to establish three-dimensional (3D) in vitro platforms that mimic the progression of liver fibrosis.

This review outlines the pathophysiology of liver fibrosis and highlights recent advancements in 3D in vitro liver models, including spheroids, organoids, assembloids, bioprinted constructs, and microfluidic systems. It further assesses their biological relevance, with particular focus on their capacity to reproduce fibrosis-related characteristics.

3D in vitro liver models offer significant advantages over conventional two-dimensional cultures. Although each model exhibits unique strengths, they collectively recapitulate key fibrotic features, such as extracellular matrix remodeling, hepatic stellate cell activation, and collagen deposition, in a physiologically relevant 3D setting. In particular, multilineage liver organoids and assembloids integrate architectural complexity with scalability, enabling deeper mechanistic insights and supporting therapeutic evaluation with improved translational relevance.

3D in vitro liver models represent a promising strategy to bridge the gap between in vitro studies and in vivo realities by faithfully replicating liver-specific architecture and microenvironments. With enhanced reproducibility through standardized protocols, these models hold great potential for advancing drug discovery and facilitating the development of personalized therapies for liver fibrosis.

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.

Cultured meat offers a sustainable alternative to animal farming, with the potential to reduce environmental impacts and improve food security. However, recapitulating natural meat marbling remains a significant challenge. This study presents a straightforward technology for achieving precise marbling patterns in large-scale cultured meat using self-healing hydrogels containing boronic acid-conjugated chitosan. Unlike conventional hydrogels, which require nonphysiological conditions for strong, reversible bonding, our system achieves robust reversible bonding at neutral pH through a unique mechanism: the nucleophilic groups of chitosan facilitate boronic acid-diol bond formation, exhibiting half the strength of a typical covalent bond, as demonstrated by nanomechanics analysis. The hydrogels form dual reversible networks of boronic acid-diol and hydrogen bonds, enabling self-healing and tunable stiffness. Biocompatibility studies confirm that they support the growth of mouse-derived cells and bovine-derived primary muscle cells. Each hydrogel variant optimizes mechanotransduction for the distinct requirements of fat or muscle cell culture and differentiation. This self-healing scaffolding technology enables the seamless assembly of muscle and fat monocultures into centimeter-thick meat with micrometer-scale marbling patterns, tailoring organoleptic properties and nutritional profiles without the need for meat glues or processing equipment.

Extracellular vesicles (EVs) hold significant promise for the prevention and treatment of various diseases. However, the translation of EV-based therapies into clinical practice faces considerable challenges, particularly in terms of production yield and therapeutic efficacy. Recent studies have emphasized the heterogeneity of EVs and the influence of parental cell microenvironmental signals on their biogenesis, cargo composition, and therapeutic outcomes. This review offers a comprehensive overview of strategies to optimize the therapeutic efficacy of EVs through physical, biochemical, and mechanical modulation. Additionally, it explores how microenvironmental signals affect EV cargoes and the mechanisms by which these signals can improve therapeutic efficacy. The review also addresses current challenges and potential solutions to accelerate the clinical translation of EV therapies. Ultimately, it highlights the potential of microenvironmental modulation in unlocking the full therapeutic capacity of EVs, providing key insights into their production and clinical use for treating various diseases.

Cellular biomechanics plays a critical role in cancer metastasis and tumor progression. Existing studies on cancer cell biomechanics are mostly conducted in flat 2D conditions, where cells' behavior can differ considerably from those in 3D physiological environments. Despite great advances in developing 3D in vitro models, probing cellular elasticity in 3D conditions remains a major challenge for existing technologies. In this work, optical Brillouin microscopy is utilized to longitudinally acquire mechanical images of growing cancerous spheroids over the period of 8 days. The dense mechanical mapping from Brillouin microscopy enables us to extract spatially resolved and temporally evolving mechanical features that were previously inaccessible. Using an established machine learning algorithm, it is demonstrated that incorporating these extracted mechanical features significantly improves the classification accuracy of cancer cells, from 74% to 95%. Building on this finding, a deep learning pipeline capable of accurately differentiating cancerous spheroids from normal ones solely using Brillouin images have been developed, suggesting the mechanical features of cancer cells can potentially serve as a new biomarker in cancer classification and detection.

Microfluidic devices are used for various applications in biology and medicine. From on-chip modelling of human organs for drug screening and fast and straightforward point-of-care (POC) detection of diseases to sensitive biochemical analysis, these devices can be custom-engineered using low-cost techniques. The microchannel interface is essential for these applications, as it is the interface of immobilised biomolecules that promote cell capture, attachment and proliferation, sense analytes and metabolites or provide enzymatic reaction readouts. However, common microfluidic materials do not facilitate the stable immobilisation of biomolecules required for relevant applications, making interfacial engineering necessary to attach biomolecules to the microfluidic surfaces. Interfacial engineering is performed through various immobilisation mechanisms and surface treatment techniques, which suitably modify the surface properties like chemistry and energy to obtain robust biomolecule immobilisation and long-term storage stability suitable for the final application. In this review, we provide an overview of the status of interfacial engineering in microfluidic devices, covering applications, the role of biomolecules, their immobilisation pathways and the influence of microfluidic materials. We then propose treatment techniques to optimise performance for various biological and medical applications and highlight future areas of development.

Tendon injuries present substantial challenges in clinical settings, where traditional treatments often lead to suboptimal outcomes, including scar tissue formation, reduced strength, limited range of motion, and re-ruptures. These difficulties primarily arise from the complex hierarchical structure of tendons and their limited healing capacity. Tissue engineering offers promising solutions for tendon regeneration and muscle-to-bone reconnection, typically through the use of biodegradable scaffolds that mimic the extracellular matrix of tendons, thereby supporting cell growth and tissue formation. However, several obstacles must be addressed to develop tendon reconstruction strategies suitable for clinical application. In this study, we developed computational models to design and produce Melt Electrowriting (MEW)-3D tubular structures that replicate the mechanical properties of native mouse Achilles tendons, improving the guidance of tenocyte growth and differentiation. These models incorporated a new approach to consider the non-continuum nature of printed scaffolds formed by sets of fibers interacting with one another. Moreover, these structures facilitated cell confinement, expansion, and alignment, resulting in bundle-like formations with enhanced tensile properties. Importantly, tenocyte differentiation was achieved through a two-step cell culture protocol, which involved transitioning cells from high to low serum media. This approach effectively mitigated the phenotypic drift that often occurs with long-term cell cultures. Statement of Significance Tendon injuries are a common issue in healthcare, often leading to scar tissue, weaker tendons, limited mobility, and frequent re-injury. Tendon engineering aims to address these challenges by using biodegradable "scaffolds" that mimic the natural tendon environment, supporting cell growth and tissue repair. In this study, we developed computational models to design 3D tubular structures using a printing method called Melt Electrowriting (MEW). These structures replicate the mechanical properties of mouse Achilles tendons, guiding tendon cells to grow and differentiate effectively. By creating scaffolds from interwoven fibers, we closely replicated how natural tendon fibers interact, allowing cells to form strong, organized bundles. This approach may improve engineered tendon strength and durability, addressing key challenges in tendon reconstruction strategies.

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

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

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

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

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

Peripheral nerve injuries (PNI) remain challenging to treat due to limited regeneration capacity and the lack of effective therapeutic scaffolds to support nerve repair. This study aims to develop and evaluate a 3-aminophenylboronic acid-modified hyaluronic acid (HAB) hydrogel as a 3D scaffold to enhance Dorsal root ganglion-derived stem cells (DRGSCs) attachment, migration, and neuronal differentiation for peripheral nerve regeneration. The HAB hydrogel was synthesized through an amidation reaction and characterized using Fourier transform infrared spectroscopy (FTIR) and 1H nuclear magnetic resonance (1H NMR). DRGSCs were cultured in HAB hydrogel, and neuronal differentiation was assessed through immunofluorescence staining, PCR, and multi-electrode array (MEA) recordings. Cytotoxicity, proliferation, and in vivo biocompatibility were evaluated through live/dead staining, CCK-8 assays, and subcutaneous implantation in rats. Transcriptomic analysis was performed to explore gene expression profiles. Our results shown that DRGSCs cultured in HAB hydrogel exhibited significantly improved attachment (78.5 % ± 3.2 % vs. 45.3 % ± 2.8 %, p < 0.05) and migration speeds (21.4 μm/h vs. 12.9 μm/h, p < 0.05) compared to 2D cultures. Neuronal differentiation efficiency, as indicated by Tuj1-positive cells, was also higher (72.6 % ± 4.1 % vs. 42.8 % ± 3.9 %, p < 0.01). RNA sequencing identified 990 differentially expressed genes (627 upregulated, 363 downregulated), with pathways involved in synaptic vesicle cycling, glutamatergic and GABAergic synapses significantly enriched (p < 0.05). Validation revealed that the expression trends of Gnao1 and Grm7 in the plastic petri dish and HAB hydrogel groups were consistent with the RNA sequencing results. In vivo, the hydrogel showed excellent biocompatibility, with reduced TNF-α and IL-1β expression over a 28-day degradation cycle (p < 0.01). The HAB hydrogel provides a supportive 3D microenvironment that enhances DRGSCs differentiation and electrophysiological activity, highlighting its potential as a promising scaffold for peripheral nerve regeneration and neuroregenerative medicine.

Adipose-derived stem cells (ADSCs) are mesenchymal stem cells (MSCs) derived from adipose tissue with mesenchymal lineage differentiation potential and remarkable potential in regenerative medicine. ADSCs are easily sourced from adipose tissue, share regenerative characteristics akin to other MSCs. Their convenient adherence to plastic culture flasks, coupled with their capacity for in vitro expansion and multi-lineage differentiation, underscores their promise as a robust tool for tissue repair and enhancement. The accessibility of human adipose tissue and the development of minimally invasive isolation protocols have further propelled the autologous use of ADSCs, fueling excitement in both organ repair and regenerative medicine. Consequently, research in ADSCsis experiencing rapid growth. A detailed overview of the current landscape of ADSCs isolation and differentiation capacity including the latest advancements in ADSCs usage, encompassing ongoing clinical investigations are important considerations to understand their potential to shape the landscape of regenerative medicine.

Inflammatory bowel disease (IBD) comprises chronic inflammatory conditions with complex mechanisms and diverse manifestations, posing significant clinical challenges. Traditional animal models and ethical concerns in human studies necessitate innovative approaches. This review provides an overview of human intestinal architecture in health and inflammation, emphasizing the role of microfluidics and on-chip technologies in IBD research. These technologies allow precise manipulation of cellular and microbial interactions in a physiologically relevant context, simulating the intestinal ecosystem microscopically. By integrating cellular components and replicating 3D tissue architecture, they offer promising models for studying host-microbe interactions, wound healing, and therapeutic approaches. Continuous refinement of these technologies promises to advance IBD understanding and therapy development, inspiring further innovation and cross-disciplinary collaboration.

Bone organoids offer potential for bone regeneration and in vitro organ models. Current limitations in bone organoid culture systems include low efficiency in construction and functionality, as well as increased apoptosis in prolonged cultures of larger sizes. The ketone body 3-hydroxybutyrate (3HB), synthesized in the liver, addresses these challenges effectively. Our findings suggest that 3HB increases intracellular calcium ion (Ca2+) levels in human bone marrow-derived mesenchymal stem cells (hBMSCs) by activating the hydroxycarboxylic acid receptor 2 (HCAR2). This activation initiates the cAMP/PKA/CREB pathway, which elevates the expression of anti-apoptotic genes such as myeloid cell leukemia 1 (MCL1) and B cell lymphoma 2-related protein A1 (BCL2A1), thereby reducing apoptosis. Furthermore, this pathway boosts the expression of osteogenic proteins, including Runt-related transcription factor 2 (RUNX2) and bone morphogenetic protein 2 (BMP2), facilitating osteogenesis in bone organoids. Consequently, 3HB may enhance the construction of bone organoids that are more mature, larger, and have longer viability.

The cellular organization within organoid models is important to regulate tissue specific function, yet few engineering approaches can control or direct cellular organization. Here, a photodegradable hydrogel is used to create softened regions that direct crypt formation within intestinal organoids, where the dimensions of the photosoftened regions generate predictable and defined crypt architectures. Guided by in vivo metrics of crypt morphology, this photopatterning method is used to control the width and length of in vitro organoid crypts, which ultimately defines the curvature of the epithelium. By tracking expression of differentiated Paneth cell markers in real-time, we show that epithelial curvature directs the localization of Paneth cells within engineered crypts, providing user-directed control over organoid functionality. We anticipate that our improved control over organoid architecture and thus Paneth cell localization will lead to more consistent in vitro organoid models for both mechanistic studies and translational applications.

Mesenchymal stem cells (MSCs) play a crucial role in bone formation and remodeling. Intrinsic genetic factors and extrinsic environmental cues regulate their differentiation into osteoblasts. Within the bone microenvironment, a complex network of biochemical and biomechanical signals orchestrates bone homeostasis and regeneration. In addition, the crosstalk among MSCs, immune cells, and neighboring cells-mediated by extracellular vesicles and non-coding RNAs (such as circular RNAs and micro RNAs) -profoundly influences osteogenic differentiation and bone remodeling. Recent studies have explored specific signaling pathways that contribute to effective bone regeneration, highlighting the potential of manipulating the bone microenvironment to enhance MSC functionality. The integration of advanced biomaterials, gene editing techniques, and controlled delivery systems is paving the way for more targeted and efficient regenerative therapies. Furthermore, artificial intelligence could improve bone tissue engineering, optimize biomaterial design, and enable personalized treatment strategies. This review explores the latest advancements in bone regeneration, emphasizing the intricate interplay among stem cells, immune cells, and signaling molecules. By providing a comprehensive overview of these mechanisms and their clinical implications, we aim to shed light on future research directions in this rapidly evolving field.

Trabecular titanium implants are widely used in orthopedic surgery and are known to promote osseointegration. In this study, we investigated whether primary human osteoblast-like cells grown inside a 3D trabecular titanium scaffold undergo changes in migration capacity, transcriptomic profile, and cellular phenotype as compared to the same osteoblasts not grown inside the scaffold. Scratch tests have shown that primary human osteoblast-like cells grown inside the 3D trabecular titanium scaffold promote the migration of cells from the external environment into the scaffold. Next generation sequencing analysis demonstrated that primary human osteoblast-like cells grown inside the 3D trabecular titanium scaffold modified the expression of genes involved in cell cycle and extracellular matrix remodeling, while maintaining a normal expression of the specific osteoblast markers, such as osteocalcin and osterix, as well as a comparable mineralization capacity. These data demonstrate that primary human osteoblast-like cells grown inside the titanium scaffold in a 3D environment acquire specific features favoring osseointegration.

Cerebral organoids (cORGs) obtained from induced pluripotent stem cells (iPSCs) have become significant instruments for investigating human neurophysiology, with the possibility of simulating diseases and enhancing drug discovery. The current approaches require a strict process of manual inclusion in animal-derived matrix Matrigel® and are challenged by unpredictability, operators' skill and expertise, elevated costs, and restricted scalability, impeding their extensive applicability and translational potential. In this study, we present a novel method to generate brain organoids that address these limitations. Our approach does not require a manual, operator-dependent embedding. Instead, it employs a chemically defined hydrogel in which the Matrigel® is diluted in a solution enriched with sodium alginate (SA) and sodium carboxymethylcellulose (CMC) and used as a bioink to print neural embryoid bodies (nEBs). Immunohistochemical, immunofluorescence, and gene expression analyses confirmed that SA-CMC-Matrigel® hydrogel can sustain the generation of iPSC-derived cortical cORGs as the conventional Matrigel®-based approach does. By day 40 of differentiation, hydrogel-based 3D-bioprinted cORGs showed heterogeneous and consistent masses, with a cytoarchitecture resembling an early-stage developmental fetal brain composed of neural progenitor cells PAX6+/Ki67+ organized into tubular structures, and densely packed cell somas with extensive neurites SYP+, suggestive of cortical tissue-like neuronal layer formation.

The translation of biomedical devices and drug research is an expensive and long process with a low probability of receiving FDA approval. Developing physiologically relevant in vitro models with human cells offers a solution to not only improving the odds of FDA approval but also to expand our ability to study complex in vivo systems in a simpler fashion. Animal models remain the standard for pre-clinical testing; however, the data from animal models is an unreliable extrapolation when anticipating a human response in clinical trials, thus contributing to the low rates of translation. In this review, we focus on in vitro vascular or angiogenic models because of the incremental role that the vascular system plays in the translation of biomedical research. The first section of this review discusses the most common angiogenic cytokines that are used in vitro to initiate angiogenesis, followed by angiogenic inhibitors where both initiators and inhibitors work to maintain vascular homeostasis. Next, we evaluate previously published in vitro models, where we evaluate capturing the physical environment for biomimetic in vitro modeling. These topics provide a foundation of parameters that must be considered to improve and achieve vascular biomimicry. Finally, we summarize these topics to suggest a path forward with the goal of engineering human in vitro models that emulate the in vivo environment and provide a platform for biomedical device and drug screening that produces data to support clinical translation.

Bone tumor organoids are three-dimensional cell culture models derived from patient tissues or cells, capable of highly replicating the growth patterns and cell interactions of bone tumors in vitro. Current treatments for bone tumors are hindered by challenges such as drug resistance, recurrence, and metastasis. Organoids enhance the physiological relevance of bone tumor models, thereby improving treatment precision and overcoming the limitations of current therapeutic approaches. Organoid technology has made preliminary applications in bone tumor research, including primary bone tumors, metastatic bone tumors, and bone marrow-derived bone tumors. This review will explore the establishment of bone tumor organoids, summarize their applications and prospects in various bone tumor diseases, and discuss their integration with emerging technologies. Additionally, the limitations and future directions of bone tumor organoid research will be discussed. In the future, bone tumor organoids are expected to promote the further development of precision medicine.

Early interactions between tubercle bacilli and lung cells are critical in tuberculosis (TB) pathogenesis. Conventional two-dimensional cell cultures fail to replicate the multicellular complexity of lungs. We introduce a three-dimensional pulmosphere model for Mycobacterium tuberculosis infection in bovine systems, demonstrating through comprehensive transcriptome and proteome analyses that these multicellular spheroids closely mimic lung cell diversity, interactions, and extracellular matrix (ECM) composition. Cell viability, hypoxia, and reactive oxygen species assessments over three weeks confirm the model's suitability. To establish infection, we employed M. bovis BCG-an attenuated vaccine strain, and M. tuberculosis H37Rv-a laboratory adapted human clinical strain that is attenuated for cattle infection compared to M. bovis. Both infection upregulated key host pathways; however, M. tuberculosis induced distinct responses, including enhanced ECM receptors expression, neutrophil chemotaxis, interferon signaling, and RIG-1 signaling. A six genes/protein signature- IRF1, CCL5, CXCL8, CXCL10, SERPINE1, and CFB -emerges as an early host response marker to M. tuberculosis infection. Infection with virulent M. bovis and M. orygis revealed a shared upregulated gene signature across Mycobacterium tuberculosis complex species, but with pathogen-specific variations. This study presents a robust ex vivo bovine pulmosphere TB model with implications in biomarkers discovery, high-throughput drug screening, and TB control strategies.

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.

Organoids represent a significant advancement in disease modeling, demonstrated by their capacity to mimic the physiological/pathological structure and functional characteristics of the native tissue. Recently CRISPR/Cas9 technology has emerged as a powerful tool in combination with organoids for the development of novel therapies in preclinical settings. This review explores the current literature on applications of pooled CRISPR screening in organoids and the emerging role of these models in understanding cancer. We highlight the evolution of genome-wide CRISPR gRNA library screens in organoids, noting their increasing adoption in the field over the past decade. Noteworthy studies utilizing these screens to investigate oncogenic vulnerabilities and developmental pathways in various organoid systems are discussed. Despite the promise organoids hold, challenges such as standardization, reproducibility, and the complexity of data interpretation remain. The review also addresses the ideas of assessing tumor organoids (tumoroids) against established cancer hallmarks and the potential of studying intercellular cooperation within these models. Ultimately, we propose that organoids, particularly when personalized for patient-specific applications, could revolutionize drug screening and therapeutic approaches, minimizing the reliance on traditional animal models and enhancing the precision of clinical interventions.

Cardiovascular diseases (CVD), the leading cause of death worldwide, and their strong association with fibrosis highlight the pressing need for innovative antifibrotic therapies. In vitro models have emerged as valuable tools for replicating cardiac fibrosis 'in a dish', facilitating the study of disease mechanisms and serving as platforms for drug testing and development. These in vitro systems encompass 2D and 3D models, each with its own limitations and advantages. 2D models offer high reproducibility, cost-effectiveness, and high-throughput capabilities, but they oversimplify the complex fibrotic environment. On the other hand, 3D models provide greater biological relevance but are more complex, harder to reproduce, and less suited for high-throughput screening. The choice of model depends on the specific research question and the stage of drug development. Despite significant progress, challenges remain, including the integration of immune cells in cardiac fibrosis and optimizing the scalability and throughput of highly biomimetic systems. Herein, we review recent in vitro cardiac fibrosis models, with a focus on their shared characteristics and remaining challenges, and explore how in vitro fibrosis models of other organs could inspire novel approaches in cardiac research, showcasing potential strategies that could be adapted to refine myocardial fibrosis models.

Cardiovascular disease remains the leading cause of death worldwide, yet despite massive investment in drug discovery, the progress of cardiovascular drugs from lab to clinic remains slow. It is a complex, costly pathway from drug discovery to the clinic and failure becomes more expensive as a drug progresses along this pathway. The focus has begun to shift to optimisation of in vitro culture methodologies, not only because these must be undertaken are earlier on in the drug discovery pathway, but also because the principles of the 3Rs have become embedded in national and international legislation and regulation. Numerous studies have shown myocyte cell behaviour to be much more physiologically relevant in 3D culture compared to 2D culture, highlighting the advantages of using 3D-based models, whether microfluidic or otherwise, for preclinical drug screening. This review aims to provide an overview of the challenges in cardiovascular drug discovery, the limitations of traditional routes, and the successes in the field of preclinical models for cardiovascular drug discovery. It focuses on the particular role biomimicry can play, but also the challenges around implementation within commercial drug discovery.

Interest in Current Good Manufacturing Practices (cGMP)-grade extracellular vesicles (EVs) is expanding. Some obstacles in this new but rapidly growing field include a lack of standardization and scalability. This review focuses on automated biomanufacturing of EVs in conditioned media collected from cultured mesenchymal stromal cells (MSCs). Different automated cell culture systems are discussed, including factors affecting EV quantity and quality, isolating EVs manufactured in an automated system, and validations needed. The ultimate goal when manufacturing cGMP-grade EVs is to identify a specific application and characterize the EV population in detail. This is achieved by validating every step of the process, choosing appropriate release criteria, and assuring batch-to-batch consistency. Due to the lack of standards in the field, it is critical to ensure that the cGMP-grade EVs meet FDA standards pertaining to identity, reproducibility, sterility, safety, purity, and potency. A closed-system automated bioreactor can be a valuable tool to generate cGMP-EVs in a scalable, economical, and reproducible manner.

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

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

Cellular senescence, a process that induces irreversible cell cycle arrest in response to diverse stressors, is a primary contributor to aging and age-related diseases. Currently, exposure to hydrogen peroxide is a widely used technique for establishing in vitro cellular senescence models; however, this traditional method is inconsistent, laborious, and ineffective in vivo. To overcome these limitations, we have developed a hydrogen peroxide-releasing hydrogel that can readily and controllably induce senescence in conventional 2-dimensional cell cultures as well as advanced 3-dimensional microphysiological systems. Notably, we have established 2 platforms using our hydrogen peroxide-releasing hydrogel for investigating senolytics, which is a promising innovation in anti-geronic therapy. Conclusively, our advanced model presents a highly promising tool that offers a simple, versatile, convenient, effective, and highly adaptable technique for inducing cellular senescence. This innovation not only lays a crucial foundation for future research on aging but also markedly accelerates the development of novel therapeutic strategies targeting age-related diseases.

The extracellular matrix (ECM) plays a crucial role in organoid cultures by supporting cell proliferation and differentiation. A key feature of the ECM is its mechanical influence on the surrounding cells, directly affecting their behavior. Matrigel, the most commonly used ECM, is limited by its animal-derived origin, batch variability, and uncontrollable mechanical properties, restricting its use in 3D cell-model-based mechanobiological studies. Poly(2-alkyl-2-oxazoline) (PAOx) synthetic hydrogels represent an appealing alternative because of their reproducibility and versatile chemistry, enabling tuning of hydrogel stiffness and functionalization. Here, we studied PAOx hydrogels with differing compressive moduli for their potential to support 3D cell growth. PAOx hydrogels support spheroid and organoid growth over several days without the addition of ECM components. Furthermore, we discovered intestinal organoid epithelial polarity reversion in PAOx hydrogels and demonstrate how the tunable mechanical properties of PAOx can be used to study effects on the morphology and oxygenation of live multicellular spheroids.

Phenotypic screening of cell-cell and cell-matrix interactions is critical yet challenging for drug discovery and disease modeling. In this study, a scalable 3D tissue platform was developed by co-laying extracted natural insoluble collagen fibers, mesenchymal stem cells, endothelial cells, and neural progenitor cells for phenotypic screening. Cell growth and interactions were enhanced in the co-laid platform, evident through increased cell proliferation, viability, and vascularization. Dense vascular networks rapidly formed through cell-cell and cell-matrix interactions without adding a traditionally needed growth factor set. Both in vitro and implantation studies confirmed that these blood vessels were of human origin. To evaluate the phenotypic screening of cell-cell and cell-matrix interactions, we propose a phenotype screening prototype for stem cell interactions that utilized multivariate analysis encompassing both cell-cell and cell-matrix interactions and demonstrated its effectiveness to screen vasculature formation and autism spectrum disorder (ASD) models. Using the prototype, we confirmed that collagen crosslinking, ROCK, WNT, and YAP pathways impact vasculogenesis. In addition, ASD donor-derived neural progenitor cells can be distinguished from non-ASD control donor-derived neural progenitor cells.

Although neural tissue engineering holds great therapeutic potential for multiple clinical applications, one important challenge is the development of scaffolds that provide cues required for neural tissue development. To achieve this, biomaterial systems can be leveraged to present appropriate biological, mechanical, topographical and electrical cues that could direct cell fate. In this study, a multi-layered electrode construct was engineered to be used as a platform for 3D cell encapsulation for in vitro applications. The first layer is a conductive hydrogel coating, that improves electrical conductivity from the underlying platinum electrode. The second layer is a biosynthetic hydrogel, specifically tailored to support neural development. This layered electrode construct was electrochemically characterised, and a numerical model was applied to study electrical stimuli reaching the biosynthetic hydrogel layer. The construct was shown to effectively support the growth and proliferation of encapsulated astrocytes within the biosynthetic layer, while the numerical model will enable computational experimentation for benchmarking and study validation. This highly versatile system represents a robust tool to study the influence of electrical stimuli on neural fate, as well as investigating the development of biohybrid interfaces in vitro.

Myocardial infarction remains a leading cause of death worldwide due to the heart's limited regenerative capability and the current lack of viable therapeutic solutions. Therefore, there is an urgent need to develop effective treatment options to restore cardiac function after a heart attack. Stem cell-derived cardiac cells have been extensively utilised in cardiac tissue regeneration studies. However, the use of Matrigel as a substrate for the culture and maturation of these cells has been a major limitation for the translation of this research into clinical application. Hydrogels are emerging as a promising system to overcome this problem. They are biocompatible and can provide stem cells with a supportive scaffold that mimics the extracellular matrix, which is essential for repairing damaged tissue in the myocardium after an infarction. Thus, hydrogels provide an alternative and reproducible option in addressing myocardial infarction due to their unique potential therapeutic benefits. This review explores the different types of natural and synthetic polymers used to create hydrogels and their various delivery methods, the most common being via injection and cardiac patches and other applications such as bioprinting. Many challenges remain before hydrogels can be used in a clinical setting, but they hold great promise for the future of cardiac tissue regeneration.

White adipose tissue (WAT) plays a crucial role in energy homeostasis and secretes numerous adipokines with far-reaching effects. WAT is linked to diseases such as diabetes, cardiovascular disease, and cancer. There is a high demand for suitable in vitro models to study diseases and tissue metabolism. Most of these models are covered by 2D-monolayer cultures. This study aims to evaluate the performance of different WAT models to better derive potential applications. The stability of adipocyte characteristics in spheroids and two 3D gellan gum hydrogels with ex situ lobules and 2D-monolayer culture is analyzed. First, the differentiation to achieve adipocyte-like characteristics is determined. Second, to evaluate the maintenance of differentiated ASC-based models, an adipocyte-based model, and explants over 3 weeks, viability, intracellular lipid content, perilipin A expression, adipokine, and gene expression are analyzed. Several advantages are supported using each of the models. Including, but not limited to, the strong differentiation in 2D-monolayers, the self-assembling within spheroids, the long-term stability of the stem cell-containing hydrogels, and the mature phenotype within adipocyte-containing hydrogels and the lobules. This study highlights the advantages of 3D models due to their more in vivo-like behavior and provides an overview of the different adipose cell models.