Plexin-D1, mainly expressed in endothelial and cancer cells, regulates diverse effects, suppresses endothelial cell growth, and induces cancer cell migration and proliferation. Here, we demonstrated that plexin-D1 was cleaved by proteinase on cancer cells. To examine the role of cleaved plexin-D1 in cells, Madin-Darby canine kidney (MDCK) cells overexpressing truncated plexin-D1 were cultured in Matrigel. MDCK cells expressing plexin-D1 lacking the ectodomain (plexin-D1 ΔEC) underwent apoptosis. An adhesion assay for extracellular matrix (ECM) molecules showed that plexin-D1 ΔEC-expressing MDCK cells lost their affinity for the ECM. These results suggest that plexin-D1 ΔEC blocks integrin inside-out signaling, leading to detachment from the ECM and apoptosis, so-called anoikis. By contrast, MDCK cells expressing full-length plexin-D1 or plexin-D1 lacking the cytoplasmic domain (plexin-D1 ΔIC) developed multicellular branching tubular structures in Matrigel. This morphological change was blocked in plexin-D1-expressing MDCK cells by the hepatocyte growth factor receptor (Met) loss of function or by Met inhibitors. These results suggest that plexin-D1 associates with Met through the plexin-D1 extracellular domain, and this activates Met cytoplasmic kinase activity. We therefore conclude that plexin-D1 contains distinct domains that determine the fate of cancer cells.

Intestinal organoids are valuable tools across different disciplines, from a clinical aspect to the biomedical research, providing a unique perspective on the complexity of the gastrointestinal system. They are alternatives to common cell lines as they can offer insights into architectural functionality and reduce the use of animal models. A deeper understanding of their organoid characteristics is required to harness their full potential. Despite their beneficial uses and multiple advantages, organoids have limitations that remain unaddressed. This review aims to elucidate the principal limitations of intestinal organoids, investigate structural defects such as the deficiency in a vascularized and lymphatic system, and absence of the microbiome, restrictions in mimicking the physiological gut model, including the lack of an acid-neutralizing system or a shortage of digestive enzymes, and the difficulties in their long-term maintenance and polarity accessibility. Development of innovative techniques to address these limitations will lead to improve in vivo recapitulation and pioneering further advancements in this field.

The advent of intestinal organoids, three-dimensional structures derived from stem cells, has significantly advanced the field of biology by providing robust in vitro models that closely mimic the architecture and functionality of the human intestine. These organoids, generated from induced pluripotent stem cells (iPSCs), embryonic stem cells (ESCs), or adult stem cells, possess remarkable capabilities for self-renewal, differentiation into diverse intestinal cell types, and functional recapitulation of physiological processes, including nutrient absorption, epithelial barrier integrity, and host-microbe interactions. The utility of intestinal organoids has been extensively demonstrated in disease modeling, drug screening, and personalized medicine. Notable examples include iPSC-derived organoids, which have been effectively employed to model enteric infections, and ESC-derived organoids, which have provided critical insights into fetal intestinal development. Patient-derived organoids have emerged as powerful tools for investigating personalized therapeutics and regenerative interventions for conditions such as inflammatory bowel disease (IBD), cystic fibrosis, and colorectal cancer. Preclinical studies involving transplantation of human intestinal organoids into murine models have shown promising outcomes, including functional integration, epithelial restoration, and immune system interactions. Despite these advancements, several challenges persist, particularly in achieving reproducibility, scalability, and maturation of organoids, which hinder their widespread clinical translation. Addressing these limitations requires the establishment of standardized protocols for organoid generation, culture, storage, and analysis to ensure reproducibility and comparability of findings across studies. Nevertheless, intestinal organoids hold immense promise for transforming our understanding of gastrointestinal pathophysiology, enhancing drug development pipelines, and advancing personalized medicine. By bridging the gap between preclinical research and clinical applications, these organoids represent a paradigm shift in the exploration of novel therapeutic strategies and the investigation of gut-associated diseases.

Cardioids are 3D self-organized heart organoids directly derived from induced pluripotent stem cells (hiPSCs) aggregates. The growth and culture of cardioids is either conducted in suspension culture or heavily relies on Matrigel encapsulation. Despite the significant advancements in cardioid technology, reproducibility remains a major challenge, limiting their widespread use in both basic research and translational applications. Here, for the first time, we employed synthetic, matrix metalloproteinase (MMP)-degradable polyethylene glycol (PEG)-based hydrogels to define the effect of mechanical and biochemical cues on cardioid development. Successful cardiac differentiation is demonstrated in all the hydrogel conditions, while cardioid cultured in optimized PEG hydrogel (3 wt.% PEG-2mM RGD) underwent similar morphological development and comparable tissue functions to those cultured in Matrigel. Matrix stiffness and cell adhesion motif play a critical role in cardioid development, nascent chamber formation, contractile physiology, and endothelial cell gene enrichment. More importantly, synthetic hydrogel improved the reproducibility in cardioid properties compared to traditional suspension culture and Matrigel encapsulation. Therefore, PEG-based hydrogel has the potential to be used as an alternative to Matrigel for human cardioid culture in a variety of clinical applications including cell therapy and tissue engineering.

Organoid models are powerful tools for evaluating cancer immunotherapy that provide a more accurate representation of the tumour microenvironment (TME) and immune responses than traditional models. This review focuses on the latest advancements in organoid technologies, including immune cell co-culture, 3D bioprinting, and microfluidic systems, which enhance the modelling of TME and facilitate the assessment of immune therapies such as immune checkpoint inhibitors (ICIs), CAR-T therapies, and oncolytic viruses. Although these models have great potential in personalised cancer treatment, challenges persist in immune cell diversity, long-term culture stability, and reproducibility. Future developments integrating artificial intelligence (AI), multi-omics, and high-throughput platforms are expected to improve the predictive power of organoid models and accelerate the clinical translation of immunotherapy.

For organ-on-a-chip (OoC) engineering, the use of biocompatible coatings and materials is not only recommended but essential. Extracellular matrix (ECM) components are commonly used as coatings due to their effects on cell orientation, protein expression, differentiation, and adhesion. Among the most frequently used coatings are collagen, fibronectin, and Matrigel, according to the specific cell type and intended OoC application. Additionally, materials such as polydimethylsiloxane (PDMS), thermoplastics, chitosan, and alginate serve as scaffolding components due to their biomechanical properties and biocompatibility. Here, we discuss some of the most employed coating techniques, including SAMs, dip coating, spin coating, microcontact printing, and 3D bioprinting, each offering advantages and drawbacks. Current challenges comprise enhancing biocompatibility, exploring novel materials, and improving scalability and reproducibility.

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.

The blood-brain barrier is a physiological barrier between the vascular system and the nervous system. Under healthy conditions, it restricts the passage of most biomolecules into the brain, making drug development exceedingly challenging. Conventional cell-based in vitro models provide valuable insights into certain features of the BBB. Nevertheless, these models often lack the three-dimensional structure and dynamic interactions of the surrounding microenvironment, which greatly influence cell functionality. Consequently, considerable efforts have been made to enhance in vitro models for drug development and disease research. Recently, microfluidic organ-on-a-chip systems have emerged as promising candidates to better mimic the dynamic nature of the BBB. This review provides a comprehensive overview of recent BBB-on-chip devices. The typical building blocks, chip designs, the perfusion infrastructure, and readouts used to characterize and evaluate BBB formation are presented, analyzed, and discussed in detail. STATEMENT OF SIGNIFICANCE: The blood-brain barrier (BBB) is a highly selective barrier that controls what can enter the brain. While it protects the brain from harmful substances, it also hinders the delivery of treatments for neurological diseases such as Alzheimer's and Parkinson's. Due to its complexity, studying the BBB in living organisms remains difficult. However, recent advances in "organ-on-a-chip" technology have allowed scientists to create small, engineered models that replicate the BBB. These models provide a powerful platform to study diseases and test potential drugs with greater accuracy than traditional methods. Organ-on-a-chip devices are designed to mimic the behavior of organs or tissues in the human body, offering a more realistic and controlled environment for research. This review highlights recent breakthroughs in BBB-on-a-chip technology, showing how these models enhance current research and have the potential to transform the way we study brain diseases and develop new drugs. By integrating biology and engineering, BBB-on-a-chip technology has the potential to transform neuroscience research, improve drug development, and enhance our understanding of brain disorders.

Tumor organoid models have emerged as valuable 3D in vitro systems to study cancer behavior in a physiologically relevant environment. The composition and architecture of the extracellular matrix (ECM) play critical roles in tumor organoid culture by influencing the tumor microenvironment and tumor behavior. Traditional matrices such as Matrigel and collagen, have been widely used, but their batch-to-batch variability and limited tunability hinder their reproducibility and broader applications. To address these challenges, researchers have turned to synthetic/engineered matrices and biopolymer-based matrices, which offer precise tunability, reproducibility, and chemically defined compositions. However, these matrices also present challenges of their own. In this review, we explore the significance of ECMs in tumor organoid culture, discuss the limitations of commonly used matrices, and highlight recent advancements in engineered/synthetic matrices for improved tumor organoid modeling.

Patient-derived xenografts (PDXs) can be improved by implantation of a humanized niche. Nevertheless, the overall complexity of the current protocols, as well as the use of specific biomaterials and procedures, limits the wider adoption of this approach. Here, we identify the essential minimum steps required to create the humanized scaffolds and achieve successful acute myeloid leukemia (AML) engraftment. We compared seven biomaterials, which included both published and custom-designed materials. The highest level of bone marrow niche was achieved with extracellular matrix gels and custom collagen fiber, both of which allowed for a simple non-surgical implantation. The biomaterial selection did not influence the following AML infiltration. Regarding xenotransplantation, standard intravenous administration produced the most robust engraftment, even for two out of four otherwise non-engrafting AML samples. In contrast, direct intra-scaffold xenotransplantation did not offer any advantage. In summary, we demonstrate that the combination of an injectable biomaterial for scaffold creation plus an intravenous route for AML xenotransplantation provide the most convenient and robust approach to produce AML PDX using a humanized niche.

Malignant melanoma is a highly aggressive form of skin cancer, partly driven by a subset of cancer stem cells (CSCs) with remarkable capacities for self-renewal, differentiation, and resistance to therapy. In this study, we examined how silencing three key genes-Hif1α, KLF4, and SHH-affects CSC characteristics. Using small interfering RNA (siRNA)-based approaches, we observed significant changes at both the gene and protein levels, shedding light on how these pathways influence melanoma progression. Our results demonstrated that silencing these genes reduces the stem-like features of CSCs. Notably, Hif1α silencing triggered a marked decrease in hypoxia-related gene expression, while targeting SHH led to a reduction in Gli1, a downstream effector of SHH signaling, highlighting its potential as a therapeutic target. We also observed changes in epigenetic markers such as HDAC9 and EP300, which play crucial roles in maintaining stemness and regulating gene expression. Interestingly, these interventions appeared to reprogram CSCs, pushing them toward a phenotype distinct from both traditional CSCs and non-stem cancer cells (NCSCs). Our findings emphasize the importance of targeting key signaling pathways in melanoma CSCs and underscore the value of mimicking the tumor microenvironment in experimental models. By revealing the dynamic plasticity of melanoma CSCs, this study offers fresh insights into potential therapeutic strategies, particularly using siRNA to modulate pathways associated with tumor progression and stem cell behavior.

A variety of novel peptide-grafted hydrogels, of which peptides were derived from vitronectin (PQVTRGDVFTMP) or the laminin β4 chain (PMQKMRGDVFSP), were prepared in this study. The peptide-grafted hydrogels promoted the adhesion, proliferation and colony formation of hiPSCs and maintained their pluripotency up to passage 5 under xeno-free conditions. We successfully generated RPE cells from hiPSCs using one of the most suitable xeno-free peptide-grafted hydrogels, KVN2CK (KGCGGKGG-PQVTRGDVFTMP), which was derived from vitronectin, and confirmed the effect of these hiPSC-derived RPE cells in a rat retinal degeneration model (Royal College of Surgeons (RCS) rats) via subretinal transplantation, when we investigated functional improvements in vision in RCS rats after the transplantation of hiPSC-derived RPE cells. Our novel peptide-grafted hydrogels provided a safe and robust platform for generating single-layer hiPSC-derived RPE cells under xeno-free conditions, which indicates the potential of these hydrogels for stem cell therapy for retinal degenerative diseases in the future.

The cerebrovascular basement membrane (BM) is a key component of the blood-brain barrier (BBB). The BM provides structural support for brain microvascular endothelial cells and the supporting cells of the neurovascular unit, and facilitates cell signaling through adhesion receptors, regulates the concentration of soluble factors, and serves as an additional barrier for transport. However, our understanding of the composition of BM remains incomplete. Here we analyze recent proteomic and genomic data to assess the composition of BM in human and mouse brain, and in tissue-engineered BBB models. All data sets confirm that the main components of brain BM are collagen IV a1/2, laminin, along with agrin, perlecan, and nidogen. Transcriptomic data from human BMECs suggests that the main laminin isoform is Laminin 321, while transcriptomic data from mice and proteomic data from mice and humans suggest that Laminin 521 is the predominant isoform. Transcriptomic data from iBMECs suggest that Laminin 511 is the predominant isoform. The supporting molecules agrin, perlecan, and nidogen were detected at significant levels in all studies, although only nidogen 1 was detected in the human transcriptomic data sets. No significant differences in human BM composition were observed in BMECs along the arterio-venous axis, or in comparison of healthy and AD brains.

Recent advancements in technology have significantly expanded the scope of tumor research, progressing from the study of individual cells to more intricate tissue and organ-level analyses. Tumor organoids have emerged as a highly realistic platform for investigating tumor growth, development, and their interactions with the surrounding microenvironment. However, a notable limitation of these organoids is their lack of the diverse cellular composition typically observed in actual tumors, which hinders their ability to fully replicate the complexity of the tumor microenvironment. Immune cells play a pivotal role, and tumor immunology has become a major research hotspot. Research in tumor immunology aims to elucidate how the immune system recognizes and attacks tumor cells, as well as how tumor cells evade immune surveillance. In recent years, there has been growing interest in co-culturing immune cells with tumor organoids, an approach that has yielded valuable insights into the intricate interactions between tumors and the immune system. The aim of this paper is to review and discuss the progress achieved in co-culturing tumor organoids with immune cells. By doing so, we hope to offer a new perspective and enhance our understanding of the complexity and diversity inherent in the tumor microenvironment.

Modelling peri-implantation mammalian development using the self-organising properties of stem cells is a rapidly growing field that has advanced our understanding of cell fate decisions occurring in the early embryo. Matrigel, a basement membrane matrix, is a critical substrate used in various protocols for its efficacy in promoting stem cell growth and self-organisation. However, its role in driving stem cell lineage commitment, and whether this effect is driven by biochemical or physical cues, is not currently clear. Here, we grow embryoid bodies in suspension, Matrigel and agarose, an inert polysaccharide, to attempt to decouple the physical and biochemical roles of Matrigel and better understand how it drives stem cell differentiation. We use a combination of light microscopy, quantitative PCR and immunostaining to investigate gene and protein changes in our different culture conditions. We show that stem cell aggregates in Matrigel are hindered in their ability to elongate compared with those grown in agarose or in suspension, indicating that prohibitive role in self-organisation. Aggregates in Matrigel are also driven to differentiate into endoderm, with ectoderm differentiation inhibited. Furthermore, these effects are not due to the physical presence of Matrigel, as the same effects are not witnessed in aggregates grown in agarose. Our results thus indicate that Matrigel has a significant and complex effect on the differentiation and morphology of embryoid bodies.

Irreversible progressive pulmonary diseases drastically reduce the patient quality of life, while transplantation remains the only definitive cure. Research into lung regeneration pathways holds significant potential to expand and promote the discovery of new treatment options. Polymeric biomaterials designed to replicate key tissue characteristics (i.e., biochemical composition and mechanical cues) show promise for creating environments in which to study chronic lung diseases and initiate lung tissue regeneration. In this Viewpoint, we explore how naturally derived materials can be employed alone or combined with engineered polymer systems to create advanced tissue culture platforms. Pulmonary tissue models have historically leveraged natural materials, including basement membrane extracts and a decellularized extracellular matrix, as platforms for lung regeneration studies. Here, we provide an overview of the progression of pulmonary regenerative engineering, exploring how innovations in the growing field of tissue-informed biomaterials have the potential to advance lung regeneration research by bridging the gap between biological relevance and mechanical precision.

Patient-derived xenografts and organoids were the most common patient-derived tumor models in vitro that were utilized in personalized drug screening, and the establishment rate and duration required to be improved. Patient-derived tumor-like cell clusters (PTCs) could be established within ten days for drug screening, with high establishment rate and accuracy in predicting clinical outcomes. This study aims to explore the accuracy of PTCs based on specimens obtained from percutaneous transthoracic needle biopsy (PTNB) in lung malignancy (LM) patients, and to investigate the predictors for the success of PTC culture.

This retrospective cohort study included LM patients who underwent image-guided PTNB, and the specimens were used for PTC culture, which was followed by personalized drug screening of chemotherapy and molecular targeted therapy, and the accuracy was validated by previous or further treatments. The predictors of the success of PTC culture were identified by univariable and multivariable analyses.

A total of 68 LM patients were enrolled, consisting of 57, 7, and 4 patients with non-small cell lung cancer, small cell lung cancer, and lung metastases, respectively. Pneumothorax was the predominant adverse event for PTNB, with an incidence rate of 20.6% (14/68). PTC models based on PTNB specimens were established successfully for 56 patients in 3.8 ± 2.3 days, with an 82.4% success rate. Five patients had not received treatments before or after PTC culture. PTC drug screening reveals 88.2% (45/51) overall consistency in predicting clinical outcomes. Necrotic area over half of the tumor (hazard ratio, 0.121; 95% confidence interval, 0.025-0.598; P = 0.010) was identified as the negative predictor for the success of PTC culture.

PTC culture based on PTNB specimens could be established in 82.4% of LM patients, with a high accuracy in predicting clinical outcomes. Excessive necrosis in the tumor may predict the failure of PTC culture. Image-guided PTNB targeting enhanced or fluorodeoxyglucose‌ avid regions on images might contribute to improving the success rate of PTC culture.

Cardiomyocyte manufacturing from human pluripotent stem cells is limited by the variability of differentiation efficiencies, partly attributed to the widespread use of the tumor-derived substrate Matrigel. Here, we describe a screening approach to identify fully-defined synthetic PEG hydrogels that support iPSC-derived cardiac progenitor cell (iPSC-CPC) adhesion, survival, and differentiation into iPSC-derived cardiomyocytes (iPSC-CMs). Our PEG hydrogels supported superior iPSC-CM differentiation efficiency, with a 24% increase in cTnT expression, and greater reproducibility when compared to cells cultured on Matrigel. By combining our 5-level, 3-variable full factorial screening approach with multi-variate analysis, we showed that all substrate variables manipulated here (adhesion ligand type/concentration, stiffness) had a significant influence on iPSC-CPC confluency and that iPSC-CM differentiation was significantly influenced by adhesion ligands. These results highlight the benefit of synthetic, tunable cell culture substrates and multi-variate screening studies to identify substrate formulations for a targeted cell behavior.

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.

For several decades, research has studied the influence of the extracellular matrix (ECM) mechanical properties in cell response, primarily emphasising its elasticity as the main determinant of cell and tissue behaviour. However, the ECM is not purely elastic; it is viscoelastic. ECM viscoelasticity has now emerged as a major regulator of collective cell dynamics. This review highlights recent findings on the role of ECM viscoelasticity in development and pathology.

Ovarian cancer (OC), a prevalent and severe malignancy of the female reproductive system, often presents with mild early symptoms and is therefore diagnosed at advanced stages, leading to a poor prognosis. Current chemotherapeutic treatment relies on platinum-based combinational therapy and there have been no recent breakthroughs in the development of new drugs. Advances in organoid technology offer a novel approach to study OC by simulating tumors and their microenvironment, enhancing drug screening effectiveness and accuracy, and providing a foundation for personalized therapy. In recent years, researchers have made notable advancements, successfully developing a diverse array of OC organoid models, with biobanks serving a pivotal role in enhancing their success rates and overall efficiency. The present review summarizes the advantages of organoids over other models, such as two-dimensional cell models, three-dimensional spheres and patient-derived xenograft models, as well as the application of organoids. In particular, the current review emphasizes the application of organoids in chemotherapeutic drug screening, testing and personalized treatment. The limitations and prospects of organoid technology are also discussed. The present study aimed to reveal the unique advantages of OC organoids in chemotherapeutic applications, so as to provide insights into screening and testing new drugs for OC.

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.

Spinal cord organoids (SCOs) are in vitro models that faithfully recapitulate the basic tissue architecture and cell types of the spinal cord and play a crucial role in developmental studies, disease modeling, and drug screening. Physiological cues are required for proliferation and differentiation during SCO culture. However, commonly used basement membrane matrix products, such as Matrigel®, lack tissue-specific biophysical signals. The current study utilizes decellularization process to fabricate tissue-derived hydrogel from porcine spinal cord tissue that retain intrinsic matrix components. This gel system supported an expanded neuroepithelial scale and enhanced ventral recognition patterns during SCO cultivation. Based on the characteristics of the enlarged aggregate size, a technical system for SCO cutting and subculture are proposed to improve the economic feasibility. Finally, the advantage of S-gel in maintaining neurite outgrowth are also found, which suggests its potential application in neural-related microphysiological systems.

Transitional cold atmospheric plasma (TCAP) represents a novel technique for generating plasma remotely from a primary source. It consists of a partially nonthermal ionized gas mixture containing charged and neutral particles, photons, and free radicals. In recent years, TCAP has attracted considerable attention in biomedical applications. In order to evaluate colon cancer stem cells' (CCSCs) proliferation, apoptotic induction, inflammatory response, and survival, TCAP was utilized both directly and indirectly in this study. Using argon and helium gases, TCAP was continuously delivered in two stages during the experiment. For direct state, TCAP was irradiated onto CCSCs for 3 and 5 min. In the indirect technique, Matrigel was treated with TCAP for 5 min before the introduction of cells. In vitro assays demonstrated that TCAP exposure significantly reduced the viability of CCSCs; helium gas and direct application had greater impacts than argon. Numerous investigations confirmed the induction of apoptosis, showing that the treated groups had more apoptotic cells and altered cellular structures than controls (****p < 0.0001). A substantial increase in the Bax/Bcl-2 ratio was found by analyzing the expression of the Bax and Bcl-2 genes, indicating increased susceptibility to apoptosis (*p = 0.0177 and ***p = 0.0004). The higher efficacy of the direct helium mode was further highlighted by inflammatory marker analysis, which showed a significant reduction in interleukin-6 and interleukin-8 expression in cells directly treated with TCAP-helium compared to TCAP-argon (**p = 0.0015 and ***p = 0.0007). Lastly, the proliferation test, which relies on K i-67 expression, demonstrated a noteworthy decline in all TCAP-treated groups, with the direct helium group exhibiting the most robust impact (**p = 0.0014). Overall, the findings highlight the potential of TCAP, particularly with helium, as a promising approach for selectively targeting CCSCs and providing insights into its therapeutic mechanisms for cancer treatment. TCAP, therefore, emerges as a unique therapeutic strategy with potential applications in cancer stem cell-targeted therapies.

The antiausterity strategy in anticancer drug discovery has attracted much attention as a way to exterminate cancer cells under nutrient deprived conditions which are commonly found in solid tumors. These tumors under low nutrient stress are known to be malignant and often resist conventional drug therapy. As a potential drug candidate, we focused on the meroterpenoid natural product callistrilone O which has demonstrated extremely potent antiausterity properties toward PANC-1 pancreatic carcinoma in vitro. Here, we report for the first time the total synthesis of callistrilone O in seven steps from phloroglucinol. A Friedel-Crafts-type Michael addition and an oxidative [3+2] cycloaddition with Fetizon's reagent were used to construct the molecular skeleton. The preferential cytotoxicity of callistrilone O was also evaluated with multiple starvation-resistant cancer cell lines under low nutrient conditions. Furthermore, callistrilone O was found to strongly suppress B16 melanoma tumor growth without critical toxicity in vivo. Overall, this study presents a novel anticancer agent candidate from natural products with a concise synthetic route which can be readily applied to the synthesis of derivatives.

Human induced pluripotent stem cells (hiPSCs) can give rise to multiple lineages derived from three germ layers, endoderm, mesoderm and ectoderm. Definitive endoderm (DE) cell types and tissues have great potential for regenerative medicine applications. Current hiPSC differentiation protocols focus on the addition of soluble factors; however, extracellular matrix properties are known to also play a role in dictating cell fate. Matrigel™ is the gold standard for DE differentiation, but this xenogeneic, poorly defined basement membrane extract limits the clinical translatability of DE-derived tissues. Here we present a fully defined PEG-based hydrogel substrate to support hiPSC-derived DE differentiation. We screened hydrogel formulations presenting different adhesive peptides and matrix stiffness. Our results demonstrate that presenting a short peptide, cyclic RGD, on the engineered PEG hydrogel supports the transition from undifferentiated hiPSCs to DE using a serum-free, commercially available kit. We show that increasing substrate stiffness (G' = 1.0-4.0 kPa) results in an increased linear response in DE differentiation efficiency. We also include a temporal analysis of the expression of integrin and syndecan receptors as the hiPSCs undergo specification towards DE lineage. Finally, we show that focal adhesion kinase activity regulates hiPSC growth and DE differentiation efficiency. Overall, we present a fully defined matrix as a synthetic alternative for Matrigel™ supporting DE differentiation.

Drug-induced liver injury (DILI) presents a major challenge not only in new drug development but also in post-marketing withdrawals and the safety of food, cosmetics, and chemicals. Experimental model organisms such as the rodents have been widely used for preclinical toxicological testing. However, the tension exists associated with the ethical and sustainable use of animals in part because animals do not necessarily inform the human-specific ADME (adsorption, dynamics, metabolism and elimination) profiling. To establish alternative models in humans, in vitro hepatic tissue models have been proposed, ranging from primary hepatocytes, immortal hepatocytes, to the development of new cell resources such as stem cell-derived hepatocytes. Given the evolving number of novel alternative methods, understanding possible combinations of cell sources and culture methods will be crucial to develop the context-of-use assays. This review primarily focuses on 3D liver organoid models for conducting. We will review the relevant cell sources, bioengineering methods, selection of training compounds, and biomarkers towards the rationale design of in vitro toxicology testing.

Hematological malignancies originating from blood, bone marrow, and lymph nodes include leukemia, lymphoma, and myeloma, which necessitate the use of a distinct chemotherapeutic approach. Drug resistance frequently complicates their treatment, highlighting the need for predictive tools to guide therapeutic decisions. Conventional 2D/3D cell cultures do not fully encompass in vivo criteria, and translating disease models from mice to humans proves challenging. Organ-on-a-chip technology presents an avenue to surmount genetic disparities between species, offering precise design, concurrent manipulation of various cell types, and extrapolation of data to human physiology. The development of bone-on-a-chip (BoC) systems is crucial for accurately representing the in vivo bone microenvironment, predicting drug responses for hematological cancers, mitigating drug resistance, and facilitating personalized therapeutic interventions. BoC systems for modeling hematological cancers and drug research can encompass intricate designs and integrated platforms for analyzing drug response data to simulate disease scenarios. This review provides a comprehensive examination of BoC systems applicable to modeling hematological cancers and visualizing drug responses within the intricate context of bone. It thoroughly discusses the materials pertinent to BoC systems, suitable in vitro techniques, the predictive capabilities of BoC systems in clinical settings, and their potential for commercialization.

Alzheimer's disease (AD) arises from complex multilevel interactions between genetic, epigenetic, and environmental factors. Recent studies suggest that exposure to the environmental and occupational toxicant formaldehyde (FA) may play a significant role in AD development. However, the effects of FA exposure on Aβ and tau pathologies in human neural cell 3D culture systems remain unexplored. To investigate FA's role in AD initiation, we differentiated 3D-cultured immortalized human neural progenitor ReN cells (ReNcell VM) into neurons and glial cells, followed by FA treatment. FA exposure for 12 weeks resulted in a dose-dependent increase in Aβ40, Aβ42, and phosphorylated tau levels. To further examine FA's role in AD progression, we established a 3D human neural cell culture AD model by transfecting ReN cells with AD-related mutant genes, including mutant APP and PSEN1, which recapitulate key AD pathological events. Our findings demonstrate that FA exposure significantly elevated Aβ40, Aβ42, and phosphorylated tau levels in this 3D-cultured AD model. These results suggest that FA exposure contributes to the initiation and progression of AD pathology in 3D-cultured human neural cells.

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

The engineering construction of the liver has attracted enormous attention. Organoids, as emerging miniature three-dimensional cultivation units, hold significant potential in the biomimetic simulation of liver structure and function. Despite notable successes, organoids still face limitations such as high variability and low maturity. To overcome these challenges, engineering strategies have been established to maintain organoid stability and enhance their efficacy, laying the groundwork for the development of advanced liver organoids. The present review comprehensively summarizes the construction of engineered liver organoids and their prospective applications in biomedicine. Initially, we briefly present the latest research progress on matrix materials that maintain the three-dimensional morphology of organoids. Next, we discuss the manipulative role of engineering technologies in organoid assembly. Additionally, we outline the impact of gene-level regulation on organoid growth and development. Further, we introduce the applications of liver organoids in disease modeling, drug screening and regenerative medicine. Lastly, we overview the current obstacles and forward-looking perspectives on the future of engineered liver organoids. We anticipate that ongoing innovations in engineered liver organoids will lead to significant advancements in medical applications.

Organoids have revolutionized the whole field of biology with their ability to model complex three-dimensional human organs in vitro. Intestinal organoids were especially consequential as the first successful long-term culture of intestinal stem cells, which raised hopes for translational medical applications. Despite significant contributions to basic research, challenges remain to develop intestinal organoids into clinical tools for diagnosis, prognosis, and therapy. In this review, we outline the current state of translational research involving adult stem cell and pluripotent stem cell derived intestinal organoids, highlighting the advances and limitations in disease modeling, drug-screening, personalized medicine, and stem cell therapy. Preclinical studies have demonstrated a remarkable functional recapitulation of infectious and genetic diseases, and there is mounting evidence for the reliability of intestinal organoids as a patient-specific avatar. Breakthroughs now allow the generation of structurally and cellularly complex intestinal models to better capture a wider range of intestinal pathophysiology. As the field develops and evolves, there is a need for standardized frameworks for generation, culture, storage, and analysis of intestinal organoids to ensure reproducibility, comparability, and interpretability of these preclinical and clinical studies to ultimately enable clinical translation.

Organoids as self-organized structure derived from stem cells can recapitulate the function of an organ in miniature form which have developed great potential for clinical translation, drug screening and personalized medicine. Nevertheless, the majority of patient-derived organoids (PDOs) are currently being cultured in the basement membrane matrices (BMMs), which are constrained by xenogeneic origin, batch-to-batch variability, cost, and complexity. Besides, organoid culture relies on biochemical signals provided by various growth factors in the composition of medium. We propose sodium alginate hydrogel scaffold in addition to the fibroblast conditioned medium (FCM)-enriched culture medium that is inexpensive and easily amenable to clinical applications for the culture of bladder cancer PDOs. PDOs grown in sodium alginate and FCM based medium have proliferation potential, growth rate, and gene expression that are similar to PDOs cultured in BME. According to the results, sodium alginate has substantial mechanical properties and reduces variance in early passage bladder tumor organoid cultures collected from patients. Furthermore, using FCM based medium as an alternative solution to eliminate some essential growth factors can be considered, especially for low-resource situation and develop cost effective tumor organoids.

Organoids are miniature three-dimensional (3D) organ-like structures developed from primary cells that closely mimic the key histological, functional, and molecular characteristics of their parent organs. These structures self-organize through cell-cell and cell-matrix interaction in culture. In the last decade, organoids and allied 3D culture technologies have catalyzed studies involving developmental biology, disease biology, high-throughput drug screening, personalized medicine, biomarker discovery, tissue engineering, and regenerative medicine. Many organoid systems have been generated from the gastrointestinal system, for example, intestine, stomach, liver, pancreas, or colon. Gallbladder cancer (GBC) is the most common and highly aggressive form of biliary tract cancer. GBC is rare in the west but has a high incidence in South America and India. Prolonged chronic inflammation is implicated in the pathogenesis of GBC but the driving molecular pathways leading to neoplasia are not well understood. Gallbladder cholangiocyte organoids (GCO) will facilitate the understanding of the evolution of the disease and novel therapeutic strategies. In this review, we have discussed alternative methodologies and culture conditions developed to generate GCO models, applications that these models have been subjected to and the current limitations for the use of GCOs in addressing the challenges in GBC research.

Diffuse intrinsic pontine glioma [DIPG] is a fatal pediatric disease characterized by a post-translational modification, a replacement of lysine by methionine in position 27 of the N-terminal [H3K27M] tail of histone 3 isoform-1 [H3.1] or histone 3 isoform-3 [H3.3], respectively, expressed in the DIPG-36 and DIPG-50 cells. We investigated the role of cation channels in DIPG cells for the first time and the effects of ATP-sensitive K+[KATP] and TRPV1 channel modulators.

Experiments were performed using "in vitro" cytotoxic assays combined with the patch clamp technique, RT-PCR, Western blot, and flow cytometry assays.

The most effective anti-proliferative drugs were repaglinide and glibenclamide after short and long-term incubation [6-96 h]. These drugs reduced macroscopic currents of the DIPG cells recorded in whole-cell patch clamp. Repaglinide concentration dependently enhanced the target protein H3K27ac in Western blotting after 48 h of incubation. This drug reduced cell diameter and enhanced cleaved caspase-3 in DIPG cells; total AKT/mTOR levels and phospho-mTOR were downregulated in DIPG-36.

KATP and TRPV1 channels are functionally expressed, and sulphonylureas are effective antiproliferative upregulating H3K27ac with apoptosis in DIPG cells and the sub-micromolar concentrations in DIPG-50.

Cell therapy of skeletal muscles is a promising approach for the prevention of muscular diseases and age-related muscle atrophy. However, cell transplantation to treat muscle atrophy that does not involve disease, such as sarcopenia, is considered impossible because externally injected cells rarely engraft into non-injured muscle tissue. Additionally, skeletal muscle-specific somatic stem cells, called satellite cells, lose their ability to adhere to tissue after being cultured in vitro and transforming into myoblasts. To overcome these hurdles, we explored using extracellular matrix (ECM) components to create a niche environment conducive for myoblasts during transplantation. We demonstrated that myoblasts mixed with ECM components can be engrafted into intact skeletal muscle and significantly increase muscle mass in a mouse model. These findings implicate cell transplantation therapy as a viable option for the treatment of sarcopenia. The findings will inform advancements in regenerative medicine for skeletal muscles.

Human induced pluripotent stem cell (iPSC)-derived alveolar organoids have emerged as a system to model the alveolar epithelium in homeostasis and disease. However, alveolar organoids are typically grown in Matrigel, a mouse sarcoma-derived basement membrane matrix that offers poor control over matrix properties, prompting the development of synthetic hydrogels as a Matrigel alternative. Here, we develop a two-step culture method that involves pre-aggregation of organoids in hydrogel-based microwells followed by embedding in a synthetic hydrogel that supports alveolar organoid growth, while also offering considerable control over organoid and hydrogel properties. We find that the aggregated organoids secrete their own nascent extracellular matrix (ECM) both in the microwells and upon embedding in synthetic hydrogels, which supports their growth. Thus, the synthetic hydrogels described here allow us to de-couple exogenous and nascent ECM to interrogate the role of ECM in organoid formation.

Matrigel®/BME®, a basement membrane-like preparation, supports long-term growth of epithelial 3D organoids from adult stem cells [T. Sato et al., Nature 459, 262-265 (2009); T. Sato et al., Gastroenterology 141, 1762-1772 (2011)]. Here, we show that interaction between Matrigel's major component laminin-111 with epithelial α6β1-integrin is crucial for this process. The outer membrane protein Invasin of Yersinia is known to activate multiple integrin-β1 complexes, including integrin α6β1. A C-terminal integrin-binding fragment of Invasin, coated on culture plates, mediated gut epithelial cell adhesion. Addition of organoid growth factors allowed multipassage expansion in 2D. Polarization, junction formation, and generation of enterocytes, goblet cells, Paneth cells, and enteroendocrine cells were stable over time. Sustained expansion of other human, mouse, and even snake epithelia was accomplished under comparable conditions. The 2D "organoid sheet" format holds advantages over the 3D "in gel" format in terms of imaging, accessibility of basal and apical domains, and automation for high-throughput screening. Invasin represents a fully defined, affordable, versatile, and animal-free complement to Matrigel®/BME®.

Intestinal epithelial cells are segregated into proliferative crypts and differentiated regions. This organization relies on specific signals, including Wnt3a, which regulates cell proliferation within crypts, and Eph/Ephrin, which dictates cell positioning along the crypt-villus axis. However, studying how the spatial distributions of these signals influences crypt-villus organization is challenging both in vitro and in vivo. Here we show that micropatterns of Wnt3a can govern the size, shape and long-range organization of crypts in vitro. By adjusting the spacing between Wnt3a ligand patterns at the microscale over large surfaces, we override endogenous Wnt3a to precisely control the distribution and long-range order of crypt-like regions in primary epithelial monolayers. Additionally, an agent-based model integrating Wnt3a/BMP feedback and Eph/Ephrin repulsion effectively replicates experimental tissue compartmentalization, crypt size, shape, and organization. This combined experimental and computational approach offers a framework to study how signaling pathways help organize intestinal epithelial tissue.

Regenerative medicine refers to medical research focusing on repairing, replacing, or regenerating damaged or diseased tissues or organs. Cardiovascular disease (CVDs) is a significant health issue globally and is the leading cause of death in many countries. According to the Centers for Disease Control and Prevention (CDC), one person dies every 34 seconds in the United States from cardiovascular diseases, and according to a World Health Organization (WHO) report, cardiovascular diseases are the leading cause of death globally, taking an estimated 17.9 million lives each year. Many conventional treatments are available using different drugs for cardiovascular diseases, but these treatments are inadequate. Stem cells and nanotechnology are promising research areas for regenerative medicine treating CVDs. Regenerative medicines are a revolutionary strategy for advancing and successfully treating various diseases, intending to control cardiovascular disorders. This review is a comprehensive study of different treatment methods for cardiovascular diseases using different types of biomaterials as regenerative medicines, the importance of different stem cells in therapeutics, the expanded role of nanotechnology in treatment, the administration of several types of stem cells, their tracking, imaging, and the final observation of clinical trials on many different levels as well as it aims to keep readers up to pace on emerging therapeutic applications of some specific organs and disorders that may improve from regenerative medicine shortly.

Triple-negative breast cancer (TNBC) is highly aggressive and lacks targeted therapies, posing a major challenge in oncology. Traditional two-dimensional (2D) cell cultures fail to capture the tumor microenvironment's complexity, whereas three-dimensional (3D) cultures provide a more accurate model of tumor biology. We developed an advanced 3D culture system for TNBC cell lines BT-20 and MDA-MB-231, enhancing the hanging-drop method with Matrigel to restore essential extracellular matrix interactions. Confocal imaging showed MDA-MB-231 cells forming clusters typical of aggressive carcinoma, while BT-20 cells organized into duct-like structures. Molecular analysis of PI3K and β-catenin target genes revealed distinct expression patterns, with PI3K overexpressed and β-catenin downregulated in 3D cultures. Moreover, β-catenin distribution in the 3D cell culture closely resembles its pattern in tissue. These findings underscore the value of 3D models in understanding TNBC progression and in supporting the exploration of novel therapeutic strategies.

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.