Traditional Two-dimensional Culture Research Articles

Effect of Liquid Marble 3D Culture System on In Vitro Maturation and Embryo Development of Prepubertal Goat Oocytes.

Suboptimal culture conditions during in vitro maturation (IVM) affect oocyte developmental competence and the viability of the resulting embryo. Three-dimensional (3D) culture systems provide a more biologically appropriate environment compared to traditional two-dimensional (2D) cultures. The aim of this study was to evaluate the effect of liquid marble (LM) microbioreactors as a 3D culture system on IVM and the subsequent embryo development of prepubertal goat oocytes. The cumulus-oocyte complexes (COCs) recovered from prepubertal goat ovaries underwent IVM in drops under oil (the 2D system and the control group) and in the 3D LM system (the LM group). After IVM, oocytes were parthenogenetically activated and cultured until the blastocyst stage. The control and LM groups showed similar rates of nuclear maturation (52.17% and 44.12%) and blastocyst formation (10.64% and 10.10%). Reactive oxygen species and glutathione levels and the density of transzonal projections (TZPs) in oocytes did not differ between groups. The LM system increased mitochondrial activity and modified the organization of these organelles in the oocyte cytoplasm compared to the control group. The LM microbioreactor demonstrated the ability to improve the mitochondrial status of the oocytes and was not harmful for oocyte IVM and subsequent embryo development. Therefore, LM could be used as a 3D cost-effective culture system for the IVM of prepubertal goat oocytes.

High-throughput microfluidic spheroid technology for early detection of colistin-induced nephrotoxicity with gradient-based analysis.

Colistin is essential for treating multidrug-resistant Gram-negative bacterial infections but has significant nephrotoxic side effects. Traditional approaches for studying colistin's nephrotoxicity are challenged by the rapid metabolism of its prodrug, colistin methanesulfonate and the difficulty of obtaining adequate plasma from critically ill patients. To address these challenges, we developed the Spheroid Nephrotoxicity Assessing Platform (SNAP), a microfluidic device that efficiently detects colistin-induced toxicity in renal proximal tubular epithelial cell (RPTEC) spheroids within 48 hours using just 200 μL of patient plasma. Our findings demonstrate that SNAP not only promotes higher expression of kidney-specific markers aquaporin-1 (AQP1) and low-density lipoprotein receptor-related protein 2 (LRP2) compared to traditional two-dimensional (2D) cultures, but also exhibits increased sensitivity to colistin, with significant toxicity detected at concentrations of 50 μg ml-1 and above. Notably, SNAP's non-invasive method did not identify nephrotoxicity in plasma from healthy donors, thereby confirming its physiological relevance and showcasing superior sensitivity over 2D cultures, which yielded false-positive results. In clinical validation, SNAP accurately identified patients at risk of colistin-induced nephrotoxicity with 100% accuracy for both early and late onset and demonstrated a 75% accuracy rate in predicting the non-occurrence of nephrotoxicity. These results underline the potential of SNAP in personalized medicine, offering a non-invasive, precise and efficient tool for the assessment of antibiotic-induced nephrotoxicity, thus enhancing the safety and efficacy of treatments against resistant bacterial infections.

The importance of 3D cell culture in drug discovery and development

Three-dimensional (3D) cell culture techniques represent a transformative advancement in biomedical research, particularly in drug discovery and development. By more closely replicating the physiological and microenvironmental conditions of in vivo tissues, 3D cell cultures enable more accurate assessments of drug efficacy, toxicity, and therapeutic potential compared to traditional two-dimensional (2D) cultures. These systems not only provide a more realistic model for preclinical testing but also allow for the study of complex cell-cell and cell-matrix interactions, which are often overlooked in 2D systems. This review provides a comprehensive examination of studies utilizing spheroids and organoids in 3D culture systems for drug screening and development. Furthermore, it highlights the critical role of these models in uncovering novel therapeutic targets, understanding disease mechanisms, and optimizing drug delivery strategies. Key challenges, such as scalability, standardization, and integration with high-throughput screening platforms, are also discussed. In conclusion, 3D cell culture techniques hold immense promise for revolutionizing the drug discovery pipeline, offering a more predictive and ethical approach to preclinical research while bridging the gap between laboratory findings and clinical outcomes.

Proteomic Analysis Reveals Cadherin, Actin, and Focal Adhesion Molecule-Mediated Formation of Cervical Cancer Spheroids.

Cancer spheroids are spherical, three-dimensional (3D), in vitro assemblies of cancer cells, which are gaining importance as a useful model in cancer behavior studies. Designed to simulate key features of the in vivo tumor microenvironment, spheroids offer reliable insights for drug screening and testing applications. We observed contrasting phenotypes in 3D cervical cancer (CC) cultures. Thus, in this study, we compared the proteomes of 3D and traditional two-dimensional (2D) cultures of CC cell lines, HeLa, SiHa, and C33A. When cultured in in-house poly-(2-hydroxyethyl methacrylate)-coated plates under conditions suitable for 3D spheroid formation, these CC cell lines yielded spheroids exhibiting different features. Proteomic analysis of cells cultured in 2D and 3D cultures revealed similar protein profiles but remarkable differences in the expression levels of some proteins. In SiHa and C33A cells, the upregulation of key proteins required for spheroid formation was insufficient for the formation of compact spheroids. In contrast, HeLa cells could form compact spheroids because they upregulated the proteins, including cadherin-binding, cytoskeleton, and adhesion proteins, necessary for spheroid formation during the remodeling process. Overall, this study unravels the mechanisms underlying the formation of spheroids in the commonly used CC cell lines.

Mouse Small Intestinal Organoid Cultures.

The intestinal epithelium is a highly dynamic and self-renewing tissue that is crucial for maintaining gut homeostasis. It can be cultured in vitro from isolated crypts to form three-dimensional (3D) intestinal organoids. These organoids have the ability to proliferate and differentiate into various epithelial cell lineages, offering a more physiologically relevant model compared to traditional two-dimensional (2D) culture systems. Mesenchymal cells, located near epithelial cells, regulate epithelial behavior through paracrine signaling and provide structural support. Building on recent advances in the biology of epithelial and mesenchymal cells, we have developed a coculture system that integrates intestinal organoids with mesenchymal cells. In this system, intestinal organoids are cultured in direct or indirect contact with mesenchymal cells, allowing for the simulation of signal exchange and interactions within the in vivo-like microenvironment. This coculture system not only preserves the 3D architecture of the organoids but also enhances their physiological relevance by introducing cellular complexity. The system is capable of long-term maintenance and is adaptable to a wide range of experimental manipulations. As such, this coculture model serves as a powerful tool for studying the interactions between the intestinal epithelium and its surrounding stroma, providing new insights into stem cell biology, tissue regeneration, and disease mechanisms. Here, we introduce the methods of mouse crypt isolation, intestinal organoid culture, and its coculture with mesenchymal cell.

Recapitulating Glioma Stem Cell Niches Using 3D Spheroid Models for Glioblastoma Research.

Glioblastoma multiforme (GBM) is among the most aggressive brain cancers, and it contains glioma stem cells (GSCs) that drive tumor initiation, progression, and recurrence. These cells resist conventional therapies, contributing to high recurrence rates in GBM patients. Developing in vitro models that mimic the tumor microenvironment (TME), particularly the GSC niche, is crucial for understanding GBM growth and therapeutic resistance. Three-dimensional (3D) spheroid models provide a more physiologically relevant approach than traditional two-dimensional (2D) cultures, recapitulating key tumor features like hypoxia, cell heterogeneity, and drug resistance. This review examines scaffold-free and scaffold-based methods for generating 3D GBM spheroids, focusing on their applications in studying the cancer stem cell niche. The discussion encompasses methods such as the hanging drop, low-adhesion plates, and magnetic levitation, alongside advancements in embedding spheroids within extracellular matrix-based hydrogels and employing 3D bioprinting to fabricate more intricate tumor models. These 3D culture systems offer substantial potential for enhancing our understanding of GBM biology and devising more effective targeted therapies.

A 3D Bioprinting Approach to Studying Retinal Müller Cells.

Background/Objectives: Bioprinting is an innovative technology in tissue engineering, enabling the creation of complex biological structures. This study aims to develop a three-dimensional (3D) bioprinted model of Müller cells (MCs) to enhance our understanding of their physiological and pathological roles in the retina. Methods: We investigated two different hydrogels for their ability to support the viability and differentiation of rMC-1 cells, an immortalized retinal cell line. Using 3D bioprinting technology, we assessed cell viability, differentiation, and functional characteristics through various assays, including live/dead assays and western blot analysis. Results: The collagen-based hydrogel significantly improved the viability of rMC-1 cells and facilitated the formation of spheroid aggregates, more accurately mimicking in vivo conditions compared to traditional two-dimensional (2D) culture systems. Moreover, 3D bioprinted MCs exhibited reduced markers of gliosis and oxidative stress compared to 2D cultures. Molecular analysis revealed decreased expression of GFAP and phosphorylated ERK in the 3D setting, indicating a less stressed cellular phenotype. Conclusions: Our findings demonstrate that 3D bioprinting technologies provide a more predictive platform for studying the biology of retinal MCs, which can help in the development of targeted therapeutic strategies for retinal diseases.

Bioactive Microgels with Tunable Microenvironment as a 3D Platform to Guide the Complex Physiology of Hepatocellular Carcinoma Spheroids.

Miniaturized three-dimensional tissue models, such as spheroids, have become a highly useful and efficient platform to investigate tumor physiology and explore the effect of chemotherapeutic efficacy over traditional two-dimensional monolayer culture, since they can provide more in-depth analysis, especially in regards to intercellular interactions and diffusion. The development of most tumor spheroids relies on the high proliferative capacity and self-aggregation behavior of tumor cells. However, it often disregards the effect of microenvironmental factors mediated by extracellular matrix, which are indispensable components of tissue structure. In this study, hepatocellular carcinoma (HCC) cells are encapsulated in bioactive microgels consisting of gelatin and hyaluronic acid designed to emulate tumor microenvironment in order to induce hepatic tumor spheroid formation. Two different subtypes of HCC's, HepG2 and Hep3B cell lines, are explored. The physicomechanical and biochemical properties of the microgels, controlled by changing the crosslinking density and polymer composition, are clearly shown to have substantial influence over the formation and spheroid formation. Moreover, the spheroids made from different cells and microgel properties display highly variable chemoresistance effects, further highlighting the importance of microenvironmental factors guiding tumor spheroid physiology.

Tissue-Engineered Three-Dimensional Platforms for Disease Modeling and Therapeutic Development.

Three-dimensional (3D) tissue-engineered models are under investigation to recapitulate tissue architecture and functionality, thereby overcoming limitations of traditional two-dimensional cultures and preclinical animal models. This review highlights recent developments in 3D platforms designed to model diseases in vitro that affect numerous tissues and organs, including cardiovascular, gastrointestinal, bone marrow, neural, reproductive, and pulmonary systems. We discuss current technologies for engineered tissue models, highlighting the advantages, limitations, and important considerations for modeling tissues and diseases. Lastly, we discuss future advancements necessary to enhance the reliability of 3D models of tissue development and disease.

3D cell culture models in research: applications to lung cancer pharmacology.

Lung cancer remains one of the leading causes of cancer-related mortality worldwide, necessitating innovative research methodologies to improve treatment outcomes and develop novel strategies. The advent of three-dimensional (3D) cell cultures has marked a significant advancement in lung cancer research, offering a more physiologically relevant model compared to traditional two-dimensional (2D) cultures. This review elucidates the various types of 3D cell culture models currently used in lung cancer pharmacology, including spheroids, organoids and engineered tissue models, having pivotal roles in enhancing our understanding of lung cancer biology, facilitating drug development, and advancing precision medicine. 3D cell culture systems mimic the complex spatial architecture and microenvironment of lung tumours, providing critical insights into the cellular and molecular mechanisms of tumour progression, metastasis and drug responses. Spheroids, derived from commercialized cell lines, effectively model the tumour microenvironment (TME), including the formation of hypoxic and nutrient gradients, crucial for evaluating the penetration and efficacy of anti-cancer therapeutics. Organoids and tumouroids, derived from primary tissues, recapitulate the heterogeneity of lung cancers and are instrumental in personalized medicine approaches, supporting the simulation of in vivo pharmacological responses in a patient-specific context. Moreover, these models have been co-cultured with various cell types and biomimicry extracellular matrix (ECM) components to further recapitulate the heterotypic cell-cell and cell-ECM interactions present within the lung TME. 3D cultures have been significantly contributing to the identification of novel therapeutic targets and the understanding of resistance mechanisms against conventional therapies. Therefore, this review summarizes the latest findings in drug research involving lung cancer 3D models, together with the common laboratory-based assays used to study drug effects. Additionally, the integration of 3D cell cultures into lung cancer drug development workflows and precision medicine is discussed. This integration is pivotal in accelerating the translation of laboratory findings into clinical applications, thereby advancing the landscape of lung cancer treatment. By closely mirroring human lung tumours, these models not only enhance our understanding of the disease but also pave the way for the development of more effective and personalized therapeutic strategies.

Innovations in 3D ovarian and follicle engineering for fertility preservation and restoration.

In-vitro maturation (IVM) is the process of cultivating early-stage follicles from the primordial to the antral stage and facilitating the maturation of oocytes outside the body within a supportive environment. This intricate procedure requires the careful coordination of various factors to replicate the natural ovarian conditions. Advanced techniques for IVM are designed to mimic the natural ovarian environment and enhance the development of follicles. Three-dimensional (3D) culture systems provide a more biologically relevant setting for follicle growth compared to traditional two-dimensional (2D) cultures. Traditional culture systems, often fail to support the complex process of follicle development effectively. However, modern engineered reproductive tissues and culture systems are making it possible to create increasingly physiological in-vitro models of folliculogenesis. These innovative methods are enabling researchers and clinicians to better replicate the dynamic and supportive environment of the ovary, thereby improving the outcomes of IVM offering new hope for fertility preservation and treatment. This paper focuses on the routine 3D culture, and innovative 3D culture of ovary and follicles, including a tissue engineering scaffolds, microfluidic (dynamic) culture system, organ-on-chip models, EVATAR system, from a clinical perspective to determine the most effective approach for achieving in-vitro maturation of follicles. These techniques provide critical support for ovarian function in various ovarian-associated disorders, including primary ovarian insufficiency (POI), premature ovarian failure (POF), ovarian cancer, and age-related infertility.

Engineering Three-Dimensional Spheroid Culture for Enrichment of Proangiogenic miRNAs in Umbilical Cord Mesenchymal Stem Cells and Promotion of Angiogenesis.

In the field of regenerative medicine, umbilical cord-derived mesenchymal stem cells (UC-MSCs) have a plausible potential. However, traditional two-dimensional (2D) culture systems remain limited in replicating the complex in vivo microenvironment. Thus, three-dimensional (3D) cultures offer a more physiologically relevant model. This study explored the impact of 3D culture conditions on the UC-MSC secretome and its ability to promote angiogenesis, both in vitro and in vivo. In this study, using two distinct methods, we successfully cultured UC-MSCs: in a monolayer (2D-UC-MSCs) and as spheroids formed in U-shaped 96-well plates (3D-UC-MSCs). The presence and expression of proangiogenic miRNAs in the conditioned media (CM) of these cultures were investigated, and differential expression patterns were explored. Particularly, the CM of 3D-UC-MSCs revealed significantly higher levels of miR-21-5p, miR-126-5p, and miR-130a-3p compared to 2D-UC-MSCs. Moreover, the CM from 3D-UC-MSCs revealed a higher effect on endothelial cell proliferation, migration, and tube formation than did the CM from 2D-UC-MSCs, indicating their proangiogenic potential. In an in vivo Matrigel plug mouse model, 3D-UC-MSCs (cells) stimulated greater vascular formation compared to 2D-UC-MSCs (cells). 3D culture of UC-MSCs' secretome improves the promotion of angiogenesis.

Alternative Strategy to Analyze In Vitro Cell Invasion of 3D Cultures.

Spheroid culture is a 3D model that provides an improved replication of the in vivo microenvironment compared to traditional two-dimensional (2D) cultures. Invasion is a cellular outcome of utmost interest in cancer biology. In this protocol, we have devised an alternative strategy for evaluating cancer cell invasion in vitro, employing heterospheroids comprised of oral squamous cell carcinoma (OSCC), cancer-associated fibroblasts (CAF), and monocytes. These heterospheroids aim to mimic the tumor microenvironment (TME), including two relevant non-neoplastic cell types alongside the cancer cells. Each cell type was labeled with vital fluorescent markers emitting in distinct wavelengths before spheroid formation. Once formed, heterospheroids were seeded onto a layer of human leiomyoma-derived extracellular matrix in the upper compartment of a microporous membrane. Invasion was assessed in the z-axis using confocal microscopy. Digital images were obtained in the corresponding fluorescent channels at 10 µm intervals, covering a depth of 90 µm in the z-axis. Analysis was performed using freeware image software by calculating the integrated fluorescence intensity in each image and fluorescence channel. This approach enables a more dynamic analysis of cell invasion patterns in a multilayered context, as well as the examination of spatial co-localization of different cell types during invasion.

A NSC-34 cell line-derived spheroid model: Potential and challenges for in vitro evaluation of neurodegeneration.

Three-dimensional (3D) spheroid models aim to bridge the gap between traditional two-dimensional (2D) cultures and the complex in vivo tissue environment. These models, created by self-clustering cells to mimic a 3D environment with surrounding extracellular framework, provide a valuable research tool. The NSC-34 cell line, generated by fusing mouse spinal cord motor neurons and neuroblastoma cells, is essential for studying neurodegenerative diseases like amyotrophic lateral sclerosis (ALS), where abnormal protein accumulation, such as TAR-DNA-binding protein 43 (TDP-43), occurs in affected nerve cells. However, NSC-34 behavior in a 3D context remains underexplored, and this study represents the first attempt to create a 3D model to determine its suitability for studying pathology. We generated NSC-34 spheroids using a nonadhesive hydrogel-based template and characterized them for 6 days. Light microscopy revealed that NSC-34 cells in 3D maintained high viability, a distinct round shape, and forming stable membrane connections. Scanning electron microscopy identified multiple tunnel-like structures, while ultrastructural analysis highlighted nuclear bending and mitochondria alterations. Using inducible GFP-TDP-43-expressing NSC-34 spheroids, we explored whether 3D structure affected TDP-43 expression, localization, and aggregation. Spheroids displayed nuclear GFP-TDP-43 expression, albeit at a reduced level compared with 2D cultures and generated both TDP-35 fragments and TDP-43 aggregates. This study sheds light on the distinctive behavior of NSC-34 in 3D culture, suggesting caution in the use of the 3D model for ALS or TDP-43 pathologies. Yet, it underscores the spheroids' potential for investigating fundamental cellular mechanisms, cell adaptation in a 3D context, future bioreactor applications, and drug penetration studies. RESEARCH HIGHLIGHTS: 3D spheroid generation: NSC-34 spheroids, developed using a hydrogel-based template, showed high viability and distinct shapes for 6 days. Structural features: advanced microscopy identified tunnel-like structures and nuclear and mitochondrial changes in the spheroids. Protein dynamics: the study observed how 3D structures impact TDP-43 behavior, with altered expression but similar aggregation patterns to 2D cultures. Research implications: this study reveals the unique behavior of NSC-34 in 3D culture, suggests a careful approach to use this model for ALS or TDP-43 pathologies, and highlights its potential in cellular mechanism research and drug testing applications.

The application of organoids in colorectal diseases.

Intestinal organoids are a three-dimensional cell culture model derived from colon or pluripotent stem cells. Intestinal organoids constructed in vitro strongly mimic the colon epithelium in cell composition, tissue architecture, and specific functions, replicating the colon epithelium in an in vitro culture environment. As an emerging biomedical technology, organoid technology has unique advantages over traditional two-dimensional culture in preserving parental gene expression and mutation, cell function, and biological characteristics. It has shown great potential in the research and treatment of colorectal diseases. Organoid technology has been widely applied in research on colorectal topics, including intestinal tumors, inflammatory bowel disease, infectious diarrhea, and intestinal injury regeneration. This review focuses on the application of organoid technology in colorectal diseases, including the basic principles and preparation methods of organoids, and explores the pathogenesis of and personalized treatment plans for various colorectal diseases to provide a valuable reference for organoid technology development and application.

Human placental organoids as a model to probe early gestation maternal immune dynamics.

During pregnancy, the human placenta establishes tolerance toward fetal allogeneic tissue, where specialized trophoblast subtypes play a complex role in local and peripheral immunomodulation. However, due to inadequate models to study the early gestation of the human placenta, each trophoblast subtype's role in modulating the maternal immune response has remained elusive. Here, we derived human placental organoids from early gestation trophoblast stem cells to (1) identify patterns of immunomodulatory protein expression by trophoblast subtype and (2) evaluate the effects of the placental organoid secretome on immune cell activation and regulation. We show that the three primary trophoblast phenotypes had distinct influences on immune cell phenotype and activation and that three-dimensional culture significantly alters trophoblast immunomodulation relative to traditional two-dimensional trophoblast culture.

A quick and innovative pipeline for producing chondrocyte-homing peptide-modified extracellular vesicles by three-dimensional dynamic culture of hADSCs spheroids to modulate the fate of remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment

BackgroundExtracellular vesicles (EVs) derived from human adipose-derived mesenchymal stem cells (hADSCs) have shown great therapeutic potential in plastic and reconstructive surgery. However, the limited production and functional molecule loading of EVs hinder their clinical translation. Traditional two-dimensional culture of hADSCs results in stemness loss and cellular senescence, which is unfavorable for the production and functional molecule loading of EVs. Recent advances in regenerative medicine advocate for the use of three-dimensional culture of hADSCs to produce EVs, as it more accurately simulates their physiological state. Moreover, the successful application of EVs in tissue engineering relies on the targeted delivery of EVs to cells within biomaterial scaffolds.Methods and ResultsThe hADSCs spheroids and hADSCs gelatin methacrylate (GelMA) microspheres are utilized to produce three-dimensional cultured EVs, corresponding to hADSCs spheroids-EVs and hADSCs microspheres-EVs respectively. hADSCs spheroids-EVs demonstrate excellent production and functional molecule loading compared with hADSCs microspheres-EVs. The upregulation of eight miRNAs (i.e. hsa-miR-486-5p, hsa-miR-423-5p, hsa-miR-92a-3p, hsa-miR-122-5p, hsa-miR-223-3p, hsa-miR-320a, hsa-miR-126-3p, and hsa-miR-25-3p) and the downregulation of hsa-miR-146b-5p within hADSCs spheroids-EVs show the potential of improving the fate of remaining ear chondrocytes and promoting cartilage formation probably through integrated regulatory mechanisms. Additionally, a quick and innovative pipeline is developed for isolating chondrocyte homing peptide-modified EVs (CHP-EVs) from three-dimensional dynamic cultures of hADSCs spheroids. CHP-EVs are produced by genetically fusing a CHP at the N-terminus of the exosomal surface protein LAMP2B. The CHP + LAMP2B-transfected hADSCs spheroids were cultured with wave motion to promote the secretion of CHP-EVs. A harvesting method is used to enable the time-dependent collection of CHP-EVs. The pipeline is easy to set up and quick to use for the isolation of CHP-EVs. Compared with nontagged EVs, CHP-EVs penetrate the biomaterial scaffolds and specifically deliver the therapeutic miRNAs to the remaining ear chondrocytes. Functionally, CHP-EVs show a major effect on promoting cell proliferation, reducing cell apoptosis and enhancing cartilage formation in remaining ear chondrocytes in the M1 macrophage-infiltrated microenvironment.ConclusionsIn summary, an innovative pipeline is developed to obtain CHP-EVs from three-dimensional dynamic culture of hADSCs spheroids. This pipeline can be customized to increase EVs production and functional molecule loading, which meets the requirements for regulating remaining ear chondrocyte fate in the M1 macrophage-infiltrated microenvironment.Graphical

Comparison of Glioblastoma Cell Culture Platforms Based on Transcriptional Similarity with Paired Tissue.

No standardized in vitro cell culture models for glioblastoma (GBM) have yet been established, excluding the traditional two-dimensional culture. GBM tumorspheres (TSs) have been highlighted as a good model platform for testing drug effects and characterizing specific features of GBM, but a detailed evaluation of their suitability and comparative performance is lacking. Here, we isolated GBM TSs and extracellular matrices (ECM) from tissues obtained from newly diagnosed IDH1 wild-type GBM patients and cultured GBM TSs on five different culture platforms: (1) ordinary TS culture liquid media (LM), (2) collagen-based three-dimensional (3D) matrix, (3) patient typical ECM-based 3D matrix, (4) patient tumor ECM-based 3D matrix, and (5) mouse brain. For evaluation, we obtained transcriptome data from all cultured GBM TSs using microarrays. The LM platform exhibited the most similar transcriptional program to paired tissues based on GBM genes, stemness- and invasiveness-related genes, transcription factor activity, and canonical signaling pathways. GBM TSs can be cultured via an easy-to-handle and cost- and time-efficient LM platform while preserving the transcriptional program of the originating tissues without supplementing the ECM or embedding it into the mouse brain. In addition to applications in basic cancer research, GBM TSs cultured in LM may also serve as patient avatars in drug screening and pre-clinical evaluation of targeted therapy and as standardized and clinically relevant models for precision medicine.

Liquid Overlay and Collagen-Based Three-Dimensional Models for In Vitro Investigation of Multiple Myeloma.

Multiple myeloma (MM) clones reside in the bone marrow (BM), which plays a role in its survival and development. The interactions between MM and their neighboring mesenchymal stromal cells (MSCs) have been shown to promote MM growth and drug resistance. However, those interactions are often missing or misrepresented in traditional two-dimensional (2D) culture models. Application of novel three-dimensional (3D) models might recapitulate the BM niche more precisely, which will offer new insights into MM progression and survival. Here, we aimed to establish two 3D models, based on MSC spheroids and collagen droplets incorporating both MM cells and MSCs with the goal of replicating the native myeloma context of the BM niche. This approach revealed that although MSCs can spontaneously assemble spheroids with altered metabolic traits, MSC spheroid culture does not support the integration of MM cells. On the contrary, collagen-droplet culture supported the growth of both cell types. In collagen, MSC proliferation was reduced, with the correlating decrease in ATP production and Ki-67 expression, which might resemble invivo conditions, rather than 2D abundance of nutrients and space. MSCs and MMs were distributed homogenously throughout the collagen droplet, with an apparent CXCL12 expression in MSCs. In addition, the response of MM cells to bortezomib was substantially reduced in collagen, indicating the importance of 3D culture in the investigation of myeloma cell behavior, as drug resistance is one of the most pertinent issues in cancer therapy.

Polydopamine-Based Resveratrol-Hyaluronidase Nanomedicine Inhibited Pancreatic Cancer Cell Invasive Phenotype in Hyaluronic Acid Enrichment Tumor Sphere Model.

The dense storm microenvironment formed by an excessively cross-linked extracellular matrix, such as hyaluronic acid and collagens, serves as a major barrier that prevents drugs from reaching the deeper tumor. Current traditional two-dimensional (2D) cultures are not capable of modeling this drug delivery barrier in vitro. Thus, tumor spheroids have become increasingly important in cancer research due to their three-dimensional structure. Currently, various methods have been developed to construct tumor spheroids. However, there are still challenges, such as lengthy construction time, complex composition of added growth factors, and high cultivation costs. To address this technical bottleneck, our study combined the GelMA hydrogel system to develop a rapid and high-yield method for tumor spheroids generation. Additionally, we proposed an evaluation scheme to assess the effects of drugs on tumor spheroids. Building on the hyaluronic acid-rich pathological tumor microenvironment, we constructed a resveratrol-loaded nano-drug delivery system with tumor stroma modulation capability and used a three-dimensional (3D) tumor sphere model to simulate in vivo tumor conditions. This process was utilized to completely evaluate the ability of the nano-drug delivery system to enhance the deep penetration of resveratrol in the tumor microenvironment, providing new insights into future oncology drug screening, efficacy assessment, and drug delivery methods.