Three-dimensional Culture Research Articles

Using Microfluidic and Conventional Platforms to Evaluate the Effects of Lanthanides on Spheroid Formation

Metastasis contributes to the increased mortality rate of cancer, but the intricate mechanisms remain unclear. Cancer cells from a primary tumor invade nearby tissues and access the lymphatic or circulatory system. If these cells manage to survive and extravasate from the vasculature into distant tissues and ultimately adapt to survive, they will proliferate and facilitate malignant tumor formation. Traditional two-dimensional (2D) cell cultures offer a rapid and convenient method for validating the efficacy of anticancer drugs within a reasonable cost range, but their utility is limited because of tumors’ high heterogeneity in vivo and spatial complexities. Three-dimensional (3D) cell cultures that mimic the physiological conditions of cancer cells in vivo have gained considerable interest. In these cultures, cells assemble into spheroids through gravity, magnetic forces, or their low-adhesion to the plates. Although these approaches address some of the limitations of 2D cultures, they often require a considerable amount of time and cost. Therefore, this study aims to enhance the effectiveness of 3D culture techniques by using microfluidic systems to provide a high-throughput and sensitive pipeline for drug screening. Using these systems, we studied the effects of lanthanide elements, which have garnered interest in cancer treatment, on spheroid formation and cell spreading. Our findings suggest that these elements alter the compactness of cell spheroids and decrease cell mobility.

Establishment of a 3D organoid culture model for the investigation of adult slow-cycling putative intestinal stem cells.

The study of intestinal stem cells is a prerequisite for the development of therapies aimed at regenerating the gut. To enable investigation of adult slow-cycling H2B-GFP-retaining putative small intestinal (SI) stem cells in vitro, we have developed a three-dimensional (3D) SI organoid culture model based on the Tet-Op histone 2 B (H2B)-green fluorescent protein (GFP) fusion protein (Tet-Op-H2B-GFP) transgenic mouse. SI crypts were isolated from 6- to 12-week-old Tet-Op-H2B-GFP transgenic mice and cultured with appropriate growth factors and an animal-derived matrix (Matrigel). For in vitro transgene expression, doxycycline was added to the culture medium for 24h. By pulse-chase experiments, H2B-GFP expression and retention were assessed through direct GFP fluorescence observations, both by confocal and fluorescence microscopy and by immunohistochemistry. The percentages of H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells persisting in organoids were determined by scoring relevant GFP-positive cells. Our results indicate that 24 h exposure to doxycycline (pulse) induced ubiquitous expression of H2B-GFP in the SI organoids. During subsequent culture, in the absence of doxycycline (chase), there was a gradual loss (due to cell division) of H2B-GFP. At 6-day chase, slow-cycling H2B-GFP-retaining putative SI stem cells and H2B-GFP-retaining Paneth cells were detected in the SI organoids. The developed culture model allows detection of slow-cycling H2B-GFP-retaining putative SI stem cells and will enable the study of self-renewal and regeneration for further characterization of these cells.

A Novel Multi-compartment Rotating Bioreactor for Improving ADSC-Spheroid Formation and its Application in Neurogenic Erectile Dysfunction.

The aim of this study was to construct a multicompartment synchronous rotating bioreactor (MCSRB) for batch-production of homogenized adipose-derived stem cell (ADSC) microspheres and treat neurogenic erectile dysfunction (ED). Firstly, an MCSRB was constructed using a centrifugal device and hinged trays. Secondly, influence factors (density, rotational speed) on the formation of ADSC-spheroids were explored. Finally, a neurogenic ED model was established to verify the effectiveness and safety of ADSC-spheroids for ED treatment. An MCSRB promoted ADSCs to gather microspheres, most of which were 90-130 μm in diameter. Supernatant from three-dimensional culture led to a significant increase in cytokine expression in ADSCs and migration rate in human umbilical vein endothelial cells (HUVECs) compared to control groups. The erectile function and pathological changes of the penis were improved in the ADSC-spheroids treatment group compared to the traditional ADSCs treatment group (p < 0.01). Efficient, batch, controlled and homogenized production of ADSC stem cell microspheres, and effective improvement of erectile dysfunction in neurogenic rats can be achieved using the MCSRB device.

Matched three-dimensional organoids and two-dimensional cell lines of melanoma brain metastases mirror response to targeted molecular therapy

Despite advances in the treatment paradigm for patients with metastatic melanoma, melanoma brain metastasis (MBM) continues to represent a significant treatment challenge. The study of MBM is limited, in part, by shortcomings in existing preclinical models. Surgically eXplanted Organoids (SXOs) are ex vivo, three-dimensional cultures prepared from primary tissue samples with minimal processing that recapitulate genotypic and phenotypic features of parent tumors without an artificial extracellular scaffold. MBM SXOs were created by a novel protocol incorporating techniques for establishing glioma and cutaneous melanoma organoids. A BRAFV600K-mutant and BRAF-wildtype MBM sample were collected directly from the operating room. Organoids were cultured in an optimized culture medium without an artificial extracellular scaffold. Concurrently, matched patient-derived cell lines were created. Organoid growth was observed within 3–4 weeks, and MBM SXOs retained histological features of the parent tissue, including pleomorphic epithelioid cells with abundant cytoplasm, large nuclei, focal melanin accumulation, and strong SOX10 positivity. After sufficient growth, organoids could be manually parcellated to increase the number of replicates. Matched SXOs and cell lines demonstrated sensitivity to BRAF and MEK inhibitors. Further study using SXOs may improve the translational relevance of preclinical studies and enable the study of the metastatic melanoma tumor microenvironment.

Characterization of MSC Growth, Differentiation, and EV Production in CNF Hydrogels Under Static and Dynamic Cultures in Hypoxic and Normoxic Conditions.

Mesenchymal stem cells (MSCs) hold immense therapeutic potential due to their regenerative and immunomodulatory properties. However, to utilize this potential, it is crucial to optimize their in vitro cultivation conditions. Three-dimensional (3D) culture methods using cell-laden hydrogels aim to mimic the physiological microenvironment in vitro, thus preserving MSC biological functionalities. Cellulosic hydrogels are particularly promising due to their biocompatibility, sustainability, and tunability in terms of chemical, morphological, and mechanical properties. This study investigated the impact of (1) two physical crosslinking scenarios for hydrogels derived from anionic cellulose nanofibers (to-CNF) used to encapsulate adipose-derived MSCs (adMSCs) and (2) physiological culture conditions on the in vitro proliferation, differentiation, and extracellular vesicle (EV) production of these adMSCs. The results revealed that additional Ca2+-mediated crosslinking, intended to complement the self-assembly and gelation of aqueous to-CNF in the adMSC cultivation medium, adversely affected both the mechanical properties of the hydrogel spheres and the growth of the encapsulated cells. However, cultivation under dynamic and hypoxic conditions significantly improved the proliferation and differentiation of the encapsulated adMSCs. Furthermore, it was demonstrated that the adMSCs in the CNF hydrogel spheres exhibited potential for scalable EV production with potent immunosuppressive capacities in a bioreactor system. These findings underscore the importance of physiological culture conditions and the suitability of cellulosic materials for enhancing the therapeutic potential of MSCs. Overall, this study provides valuable insights for optimizing the in vitro cultivation of MSCs for various applications, including tissue engineering, drug testing, and EV-based therapies.

Patient-Derived Organoids: A Game-Changer in Personalized Cancer Medicine.

Research on cancer therapies has benefited from predictive tools capable of simulating treatment response and other disease characteristics in a personalized manner, in particular three-dimensional cell culture models. Such models include tumor-derived spheroids, multicellular spheroids including organotypic multicellular spheroids, and tumor-derived organoids. Additionally, organoids can be grown from various cancer cell types, such as pluripotent stem cells and induced pluripotent stem cells, progenitor cells, and adult stem cells. Although patient-derived xenografts and genetically engineered mouse models replicate human disease in vivo, organoids are less expensive, less labor intensive, and less time-consuming, all-important aspects in high-throughput settings. Like in vivo models, organoids mimic the three-dimensional structure, cellular heterogeneity, and functions of primary tissues, with the advantage of representing the normal oxygen conditions of patient organs. In this review, we summarize the use of organoids in disease modeling, drug discovery, toxicity testing, and precision oncology. We also summarize the current clinical trials using organoids.

A Novel Hydrogel Sponge for Three-Dimensional Cell Culture

Background/Objectives: Three-dimensional (3D) cell culture technologies allow us to overcome the constraints of two-dimensional methods in different fields like biochemistry and cell biology and in pharmaceutical in vitro tests. In this study, a novel 3D hydrogel sponge scaffold, composed of a crosslinked polyacrylic acid forming a porous matrix, has been developed and characterized. Methods: The scaffold was obtained via an innovative procedure involving thermal treatment followed by a salt-leaching step on a matrix-containing polymer along with a gas-forming agent. Based on experimental design for mixtures, a series of formulations were prepared to study the effect of the three components (polyacrylic acid, NaHCO3 and NaCl) on the scaffold mechanical properties, density, swelling behavior and morphological changes. Physical appearance, surface morphology, porosity, molecular diffusion, transparency, biocompatibility and cytocompatibility were also evaluated. Results: The hydrogel scaffolds obtained show high porosity and good optical transparency and mechanical resistance. The scaffolds were successfully employed to culture several cell lines for more than 20 days. Conclusions: The developed scaffolds could be an important tool, as such or with a specific coating, to obtain a more predictive cellular response to evaluate drugs in preclinical studies or for testing chemical compounds, biocides and cosmetics, thus reducing animal testing.

Identification and Manipulation of Spermatogonial Stem Cells with the Aim of Inducing Spermatogenesis in Vitro.

Assisted reproduction techniques for infertile men with non-obstructive azoospermia require a sufficient number of functional germ cells produced in vitro. Understanding the mechanisms that allow the resumption of spermatogenesis outside the testicular environment is crucial for fertility preservation in these patients. A review of the literature was conducted using databases such as PubMed, Scopus and Web of Science, with keywords including "spermatogonial stem cell," "germ cells," "male factor infertility," and "enrichment and propagation of SSCs in vitro." Currently, two models-"in vivo" and "in vitro"-have been developed for producing haploid germ cells. The "in vivo" models include spermatogonial stem cell transplantation and testicular xenograft techniques. In contrast, the "in vitro" models consist of conventional culture systems, organ culture, and three-dimensional culture systems, all designed to induce spermatogenesis in vitro. These culture systems enable the simulation of the seminiferous epithelium in vitro, which facilitates better regulation of cell-signaling pathways that control the self-renewal and differentiation of SSCs. This review provides up-to-date information on the organization of SSCs, focusing on the identification, proliferation, and differentiation of spermatogonia in vitro.

Development and Characterization of a Three-Dimensional Organotypic In Vitro Oral Cancer Model with Four Co-Cultured Cell Types, Including Patient-Derived Cancer-Associated Fibroblasts.

Background/Objectives: Cancer organoids have emerged as a valuable tool of three-dimensional (3D) cell cultures to investigate tumor heterogeneity and predict tumor behavior and treatment response. We developed a 3D organotypic culture model of oral squamous cell carcinoma (OSCC) to recapitulate the tumor-stromal interface by co-culturing four cell types, including patient-derived cancer-associated fibroblasts (PD-CAFs). Methods: A stainless-steel ring was used twice to create the horizontal positioning of the cancer stroma (adjoining normal oral mucosa connective tissue) and the OSCC layer (surrounding normal oral mucosa epithelial layer). Combined with a structured bi-layered model of the epithelial component and the underlying stroma, this protocol enabled us to construct four distinct portions mimicking the oral cancer tissue arising in the oral mucosa. Results: In this model, α-smooth muscle actin-positive PD-CAFs were localized in close proximity to the OSCC layer, suggesting a crosstalk between them. Furthermore, a linear laminin-γ2 expression was lacking at the interface between the OSCC layer and the underlying stromal layer, indicating the loss of the basement membrane-like structure. Conclusions: Since the specific 3D architecture and polarity mimicking oral cancer in vivo provides a more accurate milieu of the tumor microenvironment (TME), it could be crucial in elucidating oral cancer TME.

On the design and fabrication of nanoliter-volume hanging drop networks

Hanging drop cultures provide a favorable environment for the gentle, gel-free formation of highly uniform three-dimensional cell cultures often used in drug screening applications. Initial cell numbers can be limited, as with primary cells provided by minimally invasive biopsies. Therefore, it can be beneficial to divide cells into miniaturized arrays of hanging drops to supply a larger number of samples. Here, we present a framework for the miniaturization of hanging drop networks to nanoliter volumes. The principles of a single hanging drop are described and used to construct the fundamental equations for a microfluidic system composed of multiple connected drops. Constitutive equations for the hanging drop as a nonlinear capacitive element are derived for application in the electronic-hydraulic analogy, forming the basis for more complex, time-dependent numerical modeling of hanging drop networks. This is supplemented by traditional computational fluid dynamics simulation to provide further information about flow conditions within the wells. A fabrication protocol is presented and demonstrated for creating transparent, microscale arrays of pinned hanging drops. A custom interface, pressure-based fluidic system, and environmental chamber have been developed to support the device. Finally, fluid flow on the chip is demonstrated to align with expected behavior based on the principles derived for hanging drop networks. Challenges with the system and potential areas for improvement are discussed. This paper expands on the limited body of hanging drop network literature and provides a framework for designing, fabricating, and operating these systems at the microscale.

Efficient Treatment of Psoriasis Using Conditioned Media from Mesenchymal Stem Cell Spheroids Cultured to Produce Transforming Growth Factor-β1-Enriched Small-Sized Extracellular Vesicles.

Psoriasis is a common chronic inflammatory disease in which keratinocytes proliferate abnormally due to excessive immune action. Psoriasis can be associated with various comorbidities and has a significant impact on health-related quality of life. Although many systemic treatments, including biologic agents, have been developed, topical treatment remains the main option for psoriasis management. Consequently, there is an urgent need to develop topical treatments with minimal side effects and high efficacy. Mesenchymal stem cells (MSCs) exhibit excellent immune regulation, anti-inflammatory activities, and therapeutic effects, and MSC-derived extracellular vesicles (EVs) can serve as crucial mediators of functional transfer from MSCs. Therefore, this study aimed to develop a safe and easy-to-use emulsion cream for treating psoriasis using MSC conditioned media (CM) containing EVs. We developed an enhanced Wharton's jelly MSC (WJ-MSC) culture method through a three-dimensional (3D) culture containing exogenous transforming growth factor-β3. Using the 3D culture system, we obtained CM from WJ-MSCs, which yielded a higher EV production compared to that of conventional WJ-MSC culture methods, and investigated the effect of EV-enriched 3D-WJ-MSC-CM cream on psoriasis-related inflammation. Administration of the EV-enriched 3D-WJ-MSC-CM cream significantly reduced erythema, thickness, and scaling of skin lesions, alleviated imiquimod-induced psoriasiform lesions in mice, and ameliorated histopathological changes in mouse skin. The upregulated mRNA expression of inflammatory cytokines, including IL-17a, IL-22, IL-23, and IL-36, decreased in the lesions. In conclusion, we present here a new topical treatment for psoriasis using an MSC EV-enriched cream.

Effects of BYL-719 (alpelisib) on human breast cancer stem cells to overcome drug resistance in human breast cancer.

Breast cancer continues to be a major health concern and is currently the most commonly diagnosed cancer worldwide. Relapse, metastasis, and therapy resistance are major clinical issues that doctors need to address. We believe BYL-719, which is PI3 kinase p110а inhibitor, could also inhibit the breast cancer stem cell phenotype and epithelial-to-mesenchymal transition (EMT). In addition to the PI3K/AKT signaling pathway, BYL-719 can also inhibit essential cancer-related signaling pathways, all of which would ultimately act on the microenvironment of cancer stem cells, which is quite complicated and regulates the characteristics of tumors. These include the stemness and resistance of malignant tumors, plasticity of cancer stem cells, and anti-apoptotic features. A three-dimensional (3D) mammosphere culture method was used in vitro to culture and collect breast cancer stem cells (BCSCs). MTT, clonogenic, and cell apoptosis assays were used to detect cell viability, self-renewal, and differentiation abilities. A sphere formation assay under 3D conditions was used to detect the mammophore inhibition rate of BYL-719. The subpopulation of CD44+CD24- was detected using flow cytometry analysis while EMT biomarkers and essential signaling pathways were detected using western blotting. All the data were analyzed using GraphPad Prism 9 software. BCSC-like cells were obtained by using the 3D cell culture method in vitro. We confirmed that BYL-719 could inhibit BCSC-like cell proliferation in 3D cultures and that the stemness characteristics of BCSC-like cells were inhibited. The PI3K/AKT/mTOR signaling pathway could be inhibited by BYL-719, and the Notch, JAK-STAT and MAPK/ERK signaling pathways which have crosstalk in the tumor microenvironment (TME) are also inhibited. By comparing eribulin-resistant breast cancer cell lines, we confirmed that BYL-719 could effectively overcome drug resistance. The 3D cell culture is a novel and highly effective method for enriching BCSCs in vitro. Furthermore, the stemness and EMT of BCSCs were inhibited by BYL-719 by acting on various signaling pathways. Finally, we believe that drug resistance can be overcome by targeting the BCSCs. Conjugation of BYL-719 with other anti-neoplastic agents may be a promising treatment for this in clinic.

3D Scaffold-Based Culture System Enhances Preclinical Evaluation of Natural Killer Cell Therapy in A549 Lung Cancer Cells.

Cell-based immunotherapies have emerged as promising cancer treatment modalities, demonstrating remarkable clinical efficacy. As interest in applying immune cell-based therapies to solid tumors has gained momentum, experimental models that enable long-term monitoring and mimic clinical administration are increasingly necessary. This study explores the potential of scaffold-based cell culture technologies, specifically three-dimensional (3D) extracellular matrix (ECM)-like frameworks, as promising solutions. These frameworks facilitate unhindered immune cell growth and enable continuous cancer cell culture. The three-dimensional (3D) cell culture model was developed using tailored scaffolds for natural killer (NK) cell culture. Within this framework, A549 lung cancer cells were cocultured with NK cells, allowing real-time monitoring for up to 28 days. The expression of critical markers associated with anticancer drug resistance and epithelial-mesenchymal transition (EMT) was evaluated in cancer cells within this 3D culture context. Compared to conventional 2D monolayer cultures, this 3D scaffold-based culture revealed that solid tumor cells, specifically A549 cells, exhibited heightened resistance to anticancer drugs. Additionally, the 3D culture environment upregulated the expression of EMT markers namely vimentin, N-cadherin, and fibronectin, while NK and zEGFR-CAR-NK cells displayed anticancer effects. In the two-dimensional (2D) coculture, only zEGFR-CAR-NK cells exhibited such effects in the 3D coculture system, highlighting an intriguing inconsistency with the 2D culture model, further confirmed by in vivo experiments. This in vitro 3D cell culture model reliably predicts outcomes in NK immunotherapy experiments. Thus, it represents a valuable tool for investigating drug resistance mechanisms and assessing the efficacy of immune cell-based therapies. By bridging the gap between in vitro and in vivo investigations, this model effectively translates potential treatments into animal models and facilitates rigorous preclinical evaluations.

Intratumoral Heterogeneity and Metabolic Cross-Feeding in a Three-Dimensional Breast Cancer Culture: An In Silico Perspective.

Today, the intratumoral composition is a relevant factor associated with the progression and aggression of cancer. Although it suggests a metabolic interdependence among the subpopulations inside the tumor, a detailed map of how this interdependence contributes to the malignant phenotype is still lacking. To address this issue, we developed a systems biology approach integrating single-cell RNASeq and genome-scale metabolic reconstruction to map the metabolic cross-feeding among the subpopulations previously identified in the spheroids of MCF7 breast cancer. By calibrating our model with expression profiles and the experimental growth rate, we concluded that the reverse Warburg effect emerges as a mechanism to optimize community growth. Furthermore, through an in silico analysis, we identified lactate, alpha-ketoglutarate, and some amino acids as key metabolites whose disponibility alters the growth rate of the spheroid. Altogether, this work provides a strategy for assessing how space and intratumoral heterogeneity influence the metabolic robustness of cancer, issues suggesting that computational strategies should move toward the design of optimized treatments.

The scaffold protein IQGAP1 promotes reorientation of epithelial cell polarity at the two-cell stage for cystogenesis.

A single epithelial cell embedded in extracellular matrix (ECM) can proliferate to form an apical lumen-harboring cyst, whose formation is a fundamental step in epithelial organ development. At an early two-cell stage after cell division, the cell doublet typically displays "inverted" polarity, with apical and basolateral proteins being located to the ECM-facing and cell-cell-contacting plasma membranes, respectively. Correct cystogenesis requires polarity reorientation, a process containing apical protein endocytosis from the ECM-abutting periphery and subsequent apical vesicle delivery to a cell-cell contact site for lumen formation. Here, we show that downstream of the ECM-signal-transducer β1-integrin, Rac1, and its effector IQGAP1 promote apical protein endocytosis, contributing to polarity reorientation of mammalian epithelial Madin-Darby canine kidney (MDCK) cells at a later two-cell stage in three-dimensional culture. Rac1-GTP facilitates IQGAP1 interaction with the Rac-specific activator Tiam1, which also contributes to the endocytosis and enhances the effect of IQGAP1. These findings suggest that Tiam1 and IQGAP1 form a positive feedback loop to activate Rac1. With Rac1-GTP, IQGAP1 also binds to AP2α, an adaptor protein subunit for clathrin-mediated endocytosis; depletion of the AP2 complex impairs apical protein endocytosis in MDCK doublets. Thus, Rac1 likely participates in polarity reorientation at the two-cell stage via its interaction with IQGAP1.

8468 Development And Characterisation Of Three-Dimensional Cell Culture Models Of Adrenocortical Carcinoma

Abstract Disclosure: S. Feely: None. N. Mullen: None. P. Donlon: None. C. Hantel: None. M.C. dennedy: None. Adrenocortical carcinoma (ACC) is a rare malignancy carrying a poor prognosis and limited treatment options. Surgical resection is the only option for cure; but unsuitable for advanced disease. Mitotane is the approved medical therapy, but associated with treatment resistance and adverse effects. Newer therapies, such as immune checkpoint inhibitors, have limited success. Development of improved therapies is limited by current pre-clinical disease models. No available model reflects the tumour micro-environment. Three-dimensional (3D) cell culture models are increasingly used in cancer research (6), with a paucity of developed models in ACC. In the current study, we developed novel 3D models of ACC using a type-1 collagen matrix. We then characterised these as representative ACC disease models . 3D cell culture models of ACC were optimised by embedding ACC cell lines, MUC-1, HAC15 and H295R within a type-1 Collagen matrix at differing cell numbers. Expression of type-1 collagen in ACC primary tumours was determined using immunohistochemistry (n=4). Cell viability for each model was assessed by evaluating sytox blue staining by Flow Cytometry. Metabolic activity of ACC cells within the matrix was determined using AlamarBlue staining. Prolferative activity was assessed using Ki67. Steroidogenesis was evaluated by measuring cortisol, aldosterone and androstenedione using LC-MS/MS.. Expression of CYP11B1, CYP11B2 CYP17, and StAR was measured using RT-qPCR. Type 1 collagen is strongly expressed in ACC tumour microenvironment. MUC-1, H295R and HAC15 cells were successfully cultured in a type 1 collagen matrix. MUC-1 cells cultured in the collagen matrix remain viable throughout the period of experimentation with optimum viability between 14 and 21 days. H295R cells also remain viable with optimum viability at an earlier stage of 7-14 days. HAC15 cells demonstrate high resilience in 3D cell culture with maximum viability throughout the entire period of experimentation up to 21 days. All three models increase their metabolic and proliferative activity over time. All three steroids are expressed by all three models with an increase in aldosterone secretion observed in 3D HAC15 and H295R models compared to monolayer (p&amp;gt;0.001) at days 7 and 14. All models express key steroidogenic enzymes. 3D ACC cell culture models were developed successfully using collagen type 1 abundant in ACC. Typical characteristics of the ACC tumour environment included (i) the presence of live and necrotic cells (ii) high metabolic activity and proliferation, reflective of cell turnover.. Capacity of cells to spread and proliferate within the 3D cell culture models was limited by available collagen substrate. Steroidogenic capacity was demonstrated in 3D cell culture. This model therefore retained key characteristics of ACC and shows promise as an animal-sparing, pre-clinical model. Presentation: 6/1/2024

Alternative Method for Obtaining Human-Induced Pluripotent Stem Cell Lines and Three-Dimensional Growth: A Simplified, Passage-Free Approach that Minimizes Labor.

Induced pluripotent stem cells (iPSCs) hold significant promise for numerous applications in regenerative medicine, disease modeling, and drug discovery. However, the conventional workflow for iPSC generation, with cells grown under two-dimensional conditions, presents several challenges, including the need for specialized scientific skills such as morphologically assessing and picking colonies and removing differentiated cells during the establishment phase. Furthermore, maintaining established iPSCs in three-dimensional culture systems, while offering scalability, necessitates an enzymatic dissociation step for their further growth in a complex and time-consuming protocol. In this study, we introduce a novel approach to address these challenges by reprogramming somatic cells grown under three-dimensional conditions as spheres using a bioreactor, thereby eliminating the need for two-dimensional culture and colony picking. The iPSCs generated in this study were maintained under three-dimensional conditions simply by transferring spheres to the next bioreactor, without the need for an enzymatic dissociation step. This streamlined method simplifies the workflow, reduces technical variability and labor, and paves the way for future advancements in iPSC research and its wider applications. Key features • Establishment of induced pluripotent stem cells in a three-dimensional environment. • Maintenance and cryopreservation of iPSCs without the need for a dissociation step.

Dual scalable osteogenic microtissue engineering via GelMA microsphere-inspired mechanical training and autonomous assembling of dental pulp stem cell

Large bone tissue defects present a significant clinical challenge due to the lack of stem cells and an osteogenic microenvironment, leading to fibrotic healing and impaired bone regeneration. Microsphere-based cell-on three-dimensional (3D) culture systems show great promise for constructing osteogenic microtissues. However, the underlying mechanisms require further investigation. In this study, we propose a simple, scalable framework for highly efficient osteogenic microtissue construction, utilizing gelatin methacryloyl (GelMA) microspheres and dental pulp stem cells (DPSCs). The GelMA microspheres provide an extensive, scalable 3D framework for the autonomous adhesion, migration, and proliferation of DPSCs. Within the enormous 3D space created by the microspheres, DPSCs anchor to the microspheres and neighboring cells, inducing intrinsic tensile stress and simulating a mechanical force akin to “rock climbing training”. Transcriptomic sequencing results reveal that the 3D spatial and mechanical microenvironment modulates biological processes involved in cell adhesion, extracellular matrix organization, and the positive regulation of cell migration. Further investigations demonstrate that triggering the FAK/YAP pathway mediate mechanical driven differentiation of DPSCs into the osteoblastic lineage in the excellent osteogenic microtissues. Moreover, this simple scalable 3D framework strategy is expected to enable the efficient and large-scale preparation of stem cell-based microtissues.

High-Throughput Assessment of Metabolism-Mediated Neurotoxicity by Co-Culture of Neurospheres and Liver Spheroids.

The liver's role in the biotransformation of chemicals is critical for both augmented toxicity and detoxification. However, there has been a significant lack of effort to integrate biotransformation into in vitro neurotoxicity testing. Traditional in vitro neurotoxicity testing systems are unable to assess the qualitative and quantitative differences between parent chemicals and their metabolites as they would occur in the human body. As a result, traditional in vitro toxicity screening systems cannot incorporate hepatic biotransformation to predict the neurotoxic potential of chemical metabolites. To bridge this gap, a high-throughput, metabolism-mediated neurotoxicity testing system has been developed, which combines metabolically competent HepaRG cell spheroids with a three-dimensional (3D) culture of ReNcell VM human neural progenitor cell line. The article outlines protocols for generating HepaRG cell spheroids using an ultralow attachment (ULA) 384-well plate and for cultivating ReNcell VM in 3D on a 384-pillar plate with sidewalls and slits (384PillarPlate). Metabolically sensitive test compounds are introduced into the ULA 384-well plate containing HepaRG spheroids and then tested with 3D-cultured ReNcell VM on the 384PillarPlate. This configuration permits the in situ generation of metabolites by HepaRG cells and their subsequent testing on neurospheres. By analyzing cell viability data, researchers can determine the IC50 values for each compound, thus evaluating metabolism-mediated neurotoxicity. © 2024 Wiley Periodicals LLC. Basic Protocol 1: HepaRG spheroid culture in an ultralow attachment (ULA) 384-well plate and the assessment of drug-metabolizing enzyme (DME) activities Basic Protocol 2: 3D neural stem cell (NSC) culture on a 384PillarPlate and compound treatment for the assessment of metabolism-mediated neurotoxicity Basic Protocol 3: Image acquisition, processing, and data analysis.

Scaffold-free 3D culture systems for stem cell-based tissue regeneration.

Recent advances in scaffold-free three-dimensional (3D) culture methods have significantly enhanced the potential of stem cell-based therapies in regenerative medicine. This cutting-edge technology circumvents the use of exogenous biomaterial and prevents its associated complications. The 3D culture system preserves crucial intercellular interactions and extracellular matrix support, closely mimicking natural biological niches. Therefore, stem cells cultured in 3D formats exhibit distinct characteristics, showcasing their capabilities in promoting angiogenesis and immunomodulation. This review aims to elucidate foundational technologies and recent breakthroughs in 3D scaffold-free stem cell engineering, offering comprehensive guidance for researchers to advance this technology across various clinical applications. We first introduce the various sources of stem cells and provide a comparative analysis of two-dimensional (2D) and 3D culture systems. Given the advantages of 3D culture systems, we delve into the specific fabrication and harvesting techniques for cell sheets and spheroids. Furthermore, we explore their applications in pre-clinical studies, particularly in large animal models and clinical trials. We also discuss multidisciplinary strategies to overcome existing limitations such as insufficient efficacy, hostile microenvironments, and the need for scalability and standardization of stem cell-based products.