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Oxytocin (OXT) is a neuropeptide that has been associated with neurological diseases like autism, a strong regulating activity on anxiety and stress-related behavior, physiological effects during pregnancy and parenting, and various cellular effects in neoplastic tissue. In this study, we aimed to unravel the underlying mechanism that OXT employs to regulate cell-cell contacts, spheroid formation, and cellular migration in a 3D culture model of human MLS-402 cells. We have generated a labeled OXT receptor (OXTR) overexpressing cell line cultivated in spheroids that were treated with the OXTR agonists OXT, Atosiban, and Thr4-Gly7-oxytocin (TGOT); with or without a pre-treatment of antisense oligos (Gapmers) that induce exon skipping in the human OXTR gene. This exon skipping leads to the exclusion of exon 4 and therefore a receptor that lost its intracellular G-protein-binding domain. Sensitive digital PCR (dPCR) provided us with the means to differentiate between wild type and truncated OXTR in our cellular model. OXTR truncation differentially activated intracellular signaling cascades related to cell-cell attachment and proliferation like Akt, ERK1/2-RSK1/2, HSP27, STAT1/5, and CREB, as assessed by a Kinase Profiler Assay. Digital and transmission electron microscopy revealed increased tight junction formation and well-organized cellular protrusions into an enlarged extracellular space after OXT treatment, resulting in increased cellular survival. In summary, OXT decreases cellular migration but increases cell-cell contacts and therefore improves nutrient supply. These data reveal a novel cellular effect of OXT that might have implications for degenerating CNS diseases and tumor formation in various tissues.

The 3Rs guidelines recommend replacing animal testing with alternative models. One of the solutions proposed is organ-on-chip technology in which liver-on-chip is one of the most promising alternatives for drug screening and toxicological assays. The main challenge is to achieve the relevant in vivo-like functionalities of the liver tissue in an optimized cellular microenvironment. Here, we investigated the development of hepatic cells under dynamic conditions inside a 3D hydroscaffold embedded in a microfluidic device. The hydroscaffold is made of hyaluronic acid and composed of liver extracellular matrix components (galactosamine, collagen I/IV) with RGDS (Arg-Gly-Asp-Ser) sites for cell adhesion. The HepG2/C3A cell line was cultured under a flow rate of 10 µL/min for 21 days. After seeding, the cells formed aggregates and proliferated, forming 3D spheroids. The cell viability, functionality, and spheroid integrity were investigated and compared to static cultures. The results showed a 3D aggregate organization of the cells up to large spheroid formations, high viability and albumin production, and an enhancement of HepG2 cell functionalities. Overall, these results highlighted the role of the liver-on-chip model coupled with a hydroscaffold in the enhancement of cell functions and its potential for engineering a relevant liver model for drug screening and disease study. [ABSTRACT FROM AUTHOR]

Cancer cells adapt their intracellular energy metabolism to the oxygen-deprived tumor microenvironment (TME) to ensure tumor progression. This adaptive mechanism has focused attention on the metabolic phenotypes of tumor cells under hypoxic TME for developing novel cancer therapies. Although widely used monolayer (2D) culture does not fully reflect in vivo hypoxic TME, spheroid (3D) culture can produce a milieu similar to the TME in vivo. However, how different metabolic phenotypes are expressed in 3D cultures mimicking tumor hypoxia compared with 2D cultures under hypoxia remains unclear. To address this issue, we investigated the metabolic phenotypes of 2D- and 3D-cultured cancer cells by 13C-metabolic flux analysis (13C-MFA). Principal component analysis of 13C mass isotopomer distributions clearly demonstrated distinct metabolic phenotypes of 3D-cultured cells. 13C-MFA clarified that 3D culture significantly upregulated pyruvate carboxylase flux in line with the pyruvate carboxylase protein expression level. On the other hand, 3D culture downregulated glutaminolytic flux. Consistent with our findings, 3D-cultured cells are more resistant to a glutaminase inhibitor than 2D-cultured cells. This study suggests the importance of considering the metabolic characteristics of the particular in vitro model used for research on cancer metabolism. • Metabolic phenotype of cancer cell spheroid mimicking tumor hypoxia was studied. • 13C-Metabolic flux analysis was applied to HCT116 cells under 2D and 3D cultures. • 3D culture upregulated pyruvate anaplerosis and downregulated glutamine metabolism. • 3D culture attenuated growth inhibitory effects of glutaminase inhibitor. [ABSTRACT FROM AUTHOR]

Species differences in bilirubin glucuronidation activity are observed between humans and dogs through liver microsomes and recombinant UDP-glucuronosyltransferase 1A1. Humans exhibit higher activity than that of dogs. In this study, bilirubin glucuronidation activity was examined in canine and human primary hepatocyte spheroids formed using a 3D culture system. When spheroid development in canine and human primary hepatocytes was evaluated on days 7 and 14 after the start of culture, canine primary hepatocyte spheroids had a more distinct spherical shape than human hepatocyte spheroids, irrespective of the culture period. Furthermore, mono- and di-glucuronide generation detected in spheroids were significantly higher (P < 0.05) in human primary hepatocytes than in canine primary hepatocytes after 24 h of incubation with bilirubin for each culture period. These results suggest that there are species differences in the bilirubin glucuronidation activity of primary hepatocytes with spheroid formation between humans and dogs, with the activity being higher in humans than in dogs. [ABSTRACT FROM AUTHOR]

At present, there is an increasing need to mimic the in vivo micro-environment in the culture of cells and tissues in micro-tissue engineering. Concave microwells are becoming increasingly popular since they can provide a micro-environment that is closer to the in vivo environment compared to traditional microwells, which can facilitate the culture of cells and tissues. Here, we will summarize the fabrication methods of concave microwells, as well as their applications in micro-tissue engineering. The fabrication methods of concave microwells include traditional methods, such as lithography and etching, thermal reflow of photoresist, laser ablation, precision-computerized numerical control (CNC) milling, and emerging technologies, such as surface tension methods, the deformation of soft membranes, 3D printing, the molding of microbeads, air bubbles, and frozen droplets. The fabrication of concave microwells is transferring from professional microfabrication labs to common biochemical labs to facilitate their applications and provide convenience for users. Concave microwells have mostly been used in organ-on-a-chip models, including the formation and culture of 3D cell aggregates (spheroids, organoids, and embryoids). Researchers have also used microwells to study the influence of substrate topology on cellular behaviors. We will briefly review their applications in different aspects of micro-tissue engineering and discuss the further applications of concave microwells. We believe that building multiorgan-on-a-chip by 3D cell aggregates of different cell lines will be a popular application of concave microwells, while integrating physiologically relevant molecular analyses with the 3D culture platform will be another popular application in the near future. Furthermore, 3D cell aggregates from these biosystems will find more applications in drug screening and xenogeneic implantation. [ABSTRACT FROM AUTHOR]

Aspects related to the response of cells to photodynamic therapy (PDT) have been well studied in cell cultures, which often grow in monolayers. In this work, we propose a spheroidal model of U87MG and SKBR3 cells designed to mimic superficial tumor tissue, small spheroids (<500 µm) suitable for confocal fluorescence microscopy, and larger spheroids (>500 µm) that can be xenografted onto quail chorioallantoic membrane (CAM) to study the effects of PDT in real time. Hypericin was used as a model molecule for a hydrophobic photosensitizer that can produce singlet oxygen (1O2). 1O2 production by hypericin was detected in SKBR3 and U87MG spheroid models using a label-free technique. Vital fluorescence microscopy and flow cytometry revealed the heterogeneity of caspase-3 distribution in the cells of the spheroids. The levels of caspase-3 and apoptosis increased in the cells of spheroids 24 h after PDT. Lactate dehydrogenase activity was evaluated in the spheroids as the most reliable assay to detect differences in phototoxicity. Finally, we demonstrated the applicability of U87MG spheroids on CAM in photodiagnostics. Overall, the variability and applicability of the prepared spheroid models were demonstrated in the PDT study. [ABSTRACT FROM AUTHOR]

Nanoconstructs developed for biomedical purposes must overcome diverse biological barriers before reaching the target where playing their therapeutic or diagnostic function. In vivo models are very complex and unsuitable to distinguish the roles plaid by the multiple biological barriers on nanoparticle biodistribution and effect; in addition, they are costly, time-consuming and subject to strict ethical regulation. For these reasons, simplified in vitro models are preferred, at least for the earlier phases of the nanoconstruct development. Many in vitro models have therefore been set up. Each model has its own pros and cons: conventional 2D cell cultures are simple and cost-effective, but the information remains limited to single cells; cell monolayers allow the formation of cell–cell junctions and the assessment of nanoparticle translocation across structured barriers but they lack three-dimensionality; 3D cell culture systems are more appropriate to test in vitro nanoparticle biodistribution but they are static; finally, bioreactors and microfluidic devices can mimicking the physiological flow occurring in vivo thus providing in vitro biological barrier models suitable to reliably assess nanoparticles relocation. In this evolving context, the present review provides an overview of the most representative and performing in vitro models of biological barriers set up for nanomedical research. [ABSTRACT FROM AUTHOR]

Angiogenesis, the development of new blood vessels from pre-existing ones, is an essential process determining numerous physiological and pathological conditions. Accordingly, there is a high demand for research approaches allowing the investigation of angiogenic mechanisms and the assessment of pro- and anti-angiogenic therapeutics. The present review provides a selective overview and critical discussion of such approaches, which, in line with the 3R principle, all share the common feature that they are not based on animal experiments. They include in vitro assays to study the viability, proliferation, migration, tube formation and sprouting activity of endothelial cells in two- and three-dimensional environments, the degradation of extracellular matrix compounds as well as the impact of hemodynamic forces on blood vessel formation. These assays can be complemented by in vivo analyses of microvascular network formation in the chorioallantoic membrane assay and early stages of zebrafish larvae. In addition, the combination of experimental data and physical laws enables the mathematical modeling of tissue-specific vascularization, blood flow patterns, interstitial fluid flow as well as oxygen, nutrient and drug distribution. All these animal-free approaches markedly contribute to an improved understanding of fundamental biological mechanisms underlying angiogenesis. Hence, they do not only represent essential tools in basic science but also in early stages of drug development. Moreover, their advancement bears the great potential to analyze angiogenesis in all its complexity and, thus, to make animal experiments superfluous in the future. [ABSTRACT FROM AUTHOR]

The blood–brain barrier (BBB) is an essential structure for the maintenance of brain homeostasis. Alterations to the BBB are linked with a myriad of pathological conditions and play a significant role in the onset and evolution of neurodegenerative diseases, including Alzheimer's disease. Thus, a deeper understanding of the BBB's structure and function is mandatory for a better knowledge of neurodegenerative disorders and the development of effective therapies. Because studying the BBB in vivo imposes overwhelming difficulties, the in vitro approach remains the main possible way of research. With many in vitro BBB models having been developed over the last years, the main aim of this review is to systematically present the most relevant designs used in neurological research. In the first part of the article, the physiological and structural–functional parameters of the human BBB are detailed. Subsequently, available BBB models are presented in a comparative approach, highlighting their advantages and limitations. Finally, the new perspectives related to the study of Alzheimer's disease with the help of novel devices that mimic the in vivo human BBB milieu gives the paper significant originality. [ABSTRACT FROM AUTHOR]

Unlike mammals, teleost fish have high aromatase activity (AA) in the pituitary. However, the cells responsible for oestradiol synthesis and the local physiological roles of this hormone remain unclear. Hence, we investigated the effects of age and development on steroidogenic activity, mRNA expression, and cyp19a1b localization in the pituitary gland of the Japanese pufferfish Takifugu rubripes. Under aquaculture conditions, AA was highest after puberty, and the mRNA expression levels of cyp19a1b and the oestrogen receptors esr1 and 2b and the level of serum testosterone (T) were significantly increased after puberty compared with the other developmental stages in male and female pufferfish. Immunohistochemistry using multiple antibodies and in situ hybridization analysis revealed that Cyp19a1b colocalizes with luteinizing hormone (LH) in pituitary cells. Furthermore, Esr1 was localized in the nuclei of all hormone-producing cells, whereas Esr2b was localized only in the nuclei of Cyp19- and LH-positive cells. The administration of an aromatizable androgen (T) or oestrogen (E2) to reproductively inactive females induced LH synthesis in vivo. We prepared spheroids from pituitary cells to investigate the role of local E2 in LH synthesis in vitro. Immunohistochemical analysis of spheroids showed that T-induced LH synthesis could be blocked by an aromatase inhibitor and/or an ER antagonist but not an AR antagonist. Taken together, these findings suggest that LH synthesis is initiated in cyp19a1b-, esr1-, and esr2b-expressing cells at the onset of puberty under the control of steroidal feedback, and both feedback and local oestrogen may be involved in controlling LH synthesis in these cells. [ABSTRACT FROM AUTHOR]

Compared with traditional two-dimensional culture, a three-dimensional (3D) culture platform can not only provide more reliable prediction results, but also provide a simple, inexpensive and less time-consuming method compared with animal models. A direct in vitro model of the patient's tumor can help to achieve individualized and precise treatment. However, the existing 3D culture system based on microwell arrays has disadvantages, such as poor controllability, an uneven spheroid size, a long spheroid formation time, low-throughput and complicated operation, resulting in the need for considerable labor, etc. Here, we developed a new type of microdevice based on a 384-well plate/96-well plate microarray design. With our design, cells can quickly aggregate into clusters to form cell spheroids with better roundness. This design has the advantage of high throughput; the throughput is 33 times that of a 384-well plate. This novel microdevice is simple to process and convenient to detect without transferring the cell spheroid. The results show that the new microdevice can aggregate cells into spheroids within 24 h and can support drug and radiation sensitivity analyses in situ in approximately one week. In summary, our microdevices are fast, efficient, high-throughput, simple to process and easy to detect, providing a feasible tool for the clinical validation of individualized drug/radiation responses in patients. [ABSTRACT FROM AUTHOR]

Through the biofabrication technology using cellular aggregates (spheroids), an artificial blood vessel derived from autologous cells of human dermal fibroblast was successfully produced and is expected to be applied clinically. However, the production period remains a major problem, and the organizational construction must be optimized. It is necessary to focus on the formation and fusion of multicellular spheroids (MCS) derived from human dermal fibroblast (HDFB) and consider what affects MCS formation and fusion because these are the basis of tissue construction. In this study, we evaluated HDFB-derived MCS formation and fusion as the basis of the manufacturing process, focusing on the PI3K/AKT cascade in biofabrication technology using MCS. HDFB were used to assess the degree of MCS formation and fusion. TGF-β1 was used as an activator of the PI3K/AKT pathway, whereas LY294002 and COA-Cl were used as its inhibitors. Compared with the control group, the suppression of the PI3K/AKT pathway significantly inhibited MCS formation and fusion, whereas the activation of the PI3K/AKT pathway significantly promoted MCS formation and fusion. We identified six genes (ITGB1, PTK2, MAP2K1, PDK1, GJA1, and PI3KR1) related to cell adhesion in the PI3K/AKT pathways and confirmed a significant difference in gene expression. This study showed that the formation and fusion of HDFB-derived MCS were inhibited by suppressing the PI3K/AKT cascade, whereas it was promoted by activating the cascade with TGF-β1. This research may help elucidate the mechanism of MCS formation and fusion and optimize MCS and tissue construction, which are the basis for tissue engineering technology, and contribute to the development of regenerative medicine. [ABSTRACT FROM AUTHOR]

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies. [ABSTRACT FROM AUTHOR]

Cartilage is a connective tissue that constitutes the structure of the body and consists of chondrocytes that produce considerable collagenous extracellular matrix and plentiful ground substances, such as proteoglycan and elastin fibers. Self-repair is difficult when the cartilage is damaged because of insufficient blood supply, low cellularity, and limited progenitor cell numbers. Therefore, three-dimensional (3D) culture systems, including pellet culture, hanging droplets, liquid overlays, self-injury, and spinner culture, have attracted attention. In particular, 3D spheroid culture strategies can enhance the yield of exosome production of mesenchymal stem cells (MSCs) when compared to two-dimensional culture, and can improve cellular restorative function by enhancing the paracrine effects of MSCs. Exosomes are membrane-bound extracellular vesicles, which are intercellular communication systems that carry RNAs and proteins. Information transfer affects the phenotype of recipient cells. MSC-derived exosomes can facilitate cartilage repair by promoting chondrogenic differentiation and proliferation. In this article, we reviewed recent major advances in the application of 3D culture techniques, cartilage regeneration with stem cells using 3D spheroid culture system, the effect of exosomes on chondrogenic differentiation, and chondrogenic-specific markers related to stem cell derived exosomes. Furthermore, the utilization of MSC-derived exosomes to enhance chondrogenic differentiation for osteoarthritis is discussed. If more mechanistic studies at the molecular level are conducted, MSC-spheroid-derived exosomes will supply a better therapeutic option to improve osteoarthritis. [ABSTRACT FROM AUTHOR]

Toxicology is facing a major change in the way toxicity testing is conducted by moving away from animal experimentation towards animal-free methods. To improve the in vitro genotoxicity assessment of chemical and physical compounds, there is an urgent need to accelerate the development of 3D cell models in high-throughput DNA damage detection platforms. Among the alternative methods, hepatic cell lines are a relevant in vitro model for studying the functions of the liver. 3D HepaRG spheroids show improved hepatocyte differentiation, longevity, and functionality compared with 2D HepaRG cultures and are therefore a relevant model for predicting in vivo responses. Recently, the comet assay was developed on 3D HepaRG cells. However, this approach is still low throughput and does not meet the challenge of evaluating the toxicity and risk to humans of tens of thousands of compounds. In this study, we evaluated the performance of the high-throughput in vitro CometChip assay on 2D and 3D HepaRG cells. HepaRG cells were exposed for 48 h to several compounds (methyl methanesulfonate, etoposide, benzo[a]pyrene, cyclophosphamide, 7,12-dimethylbenz[a]anthracene, 2-acetylaminofluorene, and acrylamide) known to have different genotoxic modes of action. The resulting dose responses were quantified using benchmark dose modelling. DNA damage was observed for all compounds except 2-AAF in 2D HepaRG cells and etoposide in 3D HepaRG cells. Results indicate that the platform is capable of reliably identifying genotoxicants in 3D HepaRG cells, and provide further insights regarding specific responses of 2D and 3D models. [ABSTRACT FROM AUTHOR]

In this study, we proposed an in vitro tumor model to simulate the mechanical microenvironment and investigate the effect of compressive stress on the invasion process of malignant tumors. It has been pointed out that the biomechanical environment, as well as the biochemical environment, could affect the transformation of cancer cell migration, invasion, and metastasis. We hypothesized that the solid stress caused by the exclusion of surrounding tissue could transform tumor cells from noninvasive to invasive phenotypes. Colorectal cell spheroids were embedded and cultured in agarose gels of varying concentrations to simulate the earliest stages of tumor formation and invasion. The spheroids embedded in gels at higher concentrations showed peculiar growth after 72 h of culture, and the external compressive loading imposed on them caused peculiar growth even in the gels at lower concentrations. In conclusion, the mechanical microenvironment caused the transformation of tumor cell phenotypes, promoting the growth and invasion of tumor cell spheroids. [ABSTRACT FROM AUTHOR]

Background and Objective: Curcumin is a polyphenolic compound derived from Curcumin longa L. There is growing body of data showing the antitumor effect of curcumin in different cancers; however, its efficacy is limited by its low absorption rate. Here, we investigated the antitumor activity of turmeric oleoresin alone or in combination with paclitaxel in MCF-7 cells in monolayer cell cultures and spheroids models. Methods: The antiproliferative activities of 3 different form, curcumin, phospholipidated curcumin, and turmeric oleoresin were assessed by MTT assay. The migratory behaviors of the cells were determined by migration assay before and after treatment with curcumin. The expression levels of CyclinD1, P65, Nf-kB, and E-cadherin were studied. Findings: Curcumin suppressed cell growth in MCF-7 cells via modulation of CyclinD1 and NF-KB, which was more pronounced with turmeric oleoresin. Curcumin was able to reduce the invasiveness of MCF-7, compared to control cells through perturbation of E-cadherin. Conclusion: We demonstrated the antitumor activity of curcumin and curcumin oleoresin in breast cancer cells, supporting further investigations on the therapeutic potential of this novel anticancer agent in in vivo models. [ABSTRACT FROM AUTHOR]

Cells form hollow, spheroidal structures during the development of many tissues, including the ocular lens, inner ear, and many glands. Therefore, techniques for in vitro formation of hollow spheroids are valued for studying developmental and disease processes. Current in vitro methods require cells to self-organize into hollow morphologies; we explored an alternative strategy based on cell growth in predefined, spherical scaffolds. Our method uses sacrificial, gelatin microbeads to simultaneously template spherical chambers within a hydrogel and deliver cells into the chambers. We use mouse lens epithelial cells to demonstrate that cells can populate the internal surfaces of the chambers within a week to create numerous hollow spheroids. The platform supports manipulation of matrix mechanics, curvature, and biochemical composition to mimic in vivo microenvironments. It also provides a starting point for engineering organoids of tissues that develop from hollow spheroids.

Breast cancer is a prominent cause of death among women and is distinguished by a high occurrence of metastasis. From this perspective, apart from conventional therapies, several alternative approaches have been researched and explored in recent years, including the utilization of nano-albumin and statin medications like simvastatin. The objective of this study was to prepare albumin nanoparticles incorporating simvastatin by the self-assembly method and evaluate their impact on breast cancer metastasis and apoptosis. The data showed the prepared nanoparticles have a diameter of 185 ± 24nm and a drug loading capacity of 8.85 %. The findings exhibit improved release in a lysosomal-like environment and under acidic pH conditions. MTT data showed that nanoparticles do not exhibit a dose-dependent effect on cells. Additionally, the results from MTT, flow cytometry, and qPCR analyses demonstrated that nanoparticles have a greater inhibitory and lethal effect on MDA-MB-231 cells compared to normal simvastatin. And cause cells to accumulate in the G0/G1 phase, initiating apoptotic pathways by inhibiting cell cycle progression. Nanoparticles containing simvastatin can prevent cell invasion and migration in both monolayer and spheroid models, as compared to simvastatin alone, at microscopic levels and in gene expression. The obtained data clearly showed that, compared to simvastatin, nanoparticles containing simvastatin demonstrated significant efficacy in suppressing the growth, proliferation, invasion, and migration of cancer cells in monolayer (2D) and spheroid (3D) models., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Two-dimensional neuronal cultures have a limited ability to recapitulate the in vivo environment of the brain. Here, we introduce a three-dimensional in vitro model for human glia-to-neuron conversion, surpassing the spatial and temporal constrains of two-dimensional cultures. Focused on direct conversion to induced dopamine neurons (iDANs) relevant to Parkinson disease, the model generates functionally mature iDANs in 2 weeks and allows long-term survival. As proof of concept, we use single-nucleus RNA sequencing and molecular lineage tracing during iDAN generation and find that all glial subtypes generate neurons and that conversion relies on the coordinated expression of three neural conversion factors. We also show the formation of mature and functional iDANs over time. The model facilitates molecular investigations of the conversion process to enhance understanding of conversion outcomes and offers a system for in vitro reprogramming studies aimed at advancing alternative therapeutic strategies in the diseased brain., Competing Interests: Declaration of interests M.P. is the owner of Parmar Cells, which holds related intellectual property. M.P. performs paid consultancy to Novo Nordisk and is a member of the scientific advisory board for Arbor Biotechnologies., (Copyright © 2024 The Author(s). Published by Elsevier Inc. All rights reserved.)

Drug-induced kidney injury (DIKI) is the major cause of acute kidney injury (AKI). Renal proximal tubular epithelial cells (RPTECs) are the primary target sites of DIKI and express transporters involved in renal drug disposition. In the present study, we focused on three-dimensionally cultured human RPTECs (3D-RPTECs) with elevated expression of drug transporters to investigate their utility in DIKI evaluation. Intracellular ATP levels in 3D-RPTECs are reduced by tenofovir and cisplatin that are substrates of an organic anion transporter 1 and an organic cation transporter 2, respectively. In addition, 3D-RPTECs were exposed to 17 and 15 drugs that are positive and negative to RPTEC toxicity, respectively, for up to 28 d. The 20 % decreasing concentration of drugs for ATP amount (EC 20 ) was obtained, and the ratio of EC 20 values and clinical maximum concentration (C max ) ≤100 were used as cut-off value to evaluate potential of DIKI. The sensitivities of 3D-RPTECs were 82.4 % and 88.2 % after 7 d and 28 d of drug exposure, respectively, and the specificities were 100 % and 93.3 %, respectively. The predictive performance of 3D-RPTECs was higher than that of two-dimensional cultured RPTECs and the kidney cell line HK-2. In conclusion, 3D-RPTECs are useful for in vitro evaluation of RPTEC injury by measuring intracellular ATP levels., Competing Interests: Declaration of competing interest The authors declare the following financial interests/personal relationships which may be considered as potential competing interests: The present study was partially supported by Nikkiso Co., Ltd., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

In contrast to traditional two-dimensional cell-culture conditions, three-dimensional (3D) cell-culture models closely mimic complex in vivo conditions. However, constructing 3D cell culture models still faces challenges. In this paper, by using micro/nano fabrication method, including lithography, deposition, etching, and lift-off, we designed magnetic nanostructures resembling a crown of thorns. This magnetic crown of thorns (MCT) nanostructure enables the isolation of cells that have endocytosed magnetic particles. To assess the utility of this nanostructure, we used high-flux acquisition of Jurkat cells, an acute-leukemia cell line exhibiting the native phenotype, as an example. The novel structure enabled Jurkat cells to form spheroids within just 30 min by leveraging mild magnetic forces to bring together endocytosed magnetic particles. The size, volume, and arrangement of these spheroids were precisely regulated by the dimensions of the MCT nanostructure and the array configuration. The resulting magnetic cell clusters were uniform in size and reached saturation after 1400 s. Notably, these cell clusters could be easily separated from the MCT nanostructure through enzymatic digestion while maintaining their integrity. These clusters displayed a strong proliferation rate and survival capabilities, lasting for an impressive 96 h. Compared with existing 3D cell-culture models, the approach presented in this study offers the advantage of rapid formation of uniform spheroids that can mimic in vivo microenvironments. These findings underscore the high potential of the MCT in cell-culture models and magnetic tissue enginerring., (© 2024 IOP Publishing Ltd.)

Three-dimensional cell spheroids show promise for the reconstruction of native tissues. Herein, we report a sophisticated, uniform, and highly reproducible spheroid culture system for tissue reconstruction. A mesh-integrated culture system was designed to precisely control the uniformity and reproducibility of spheroid formation. Furthermore, we synthesized hexanoyl glycol chitosan, a material with ultralow cell adhesion properties, to further improve spheroid formation efficiency and biological function. Our results demonstrate improved biological function in various types of cells and ability to generate spheroids with complex structures composed of multiple cell types. In conclusion, our spheroid culture system offers a highly effective and widely applicable approach to generating customized spheroids with desired structural and biological features for a variety of biomedical applications., (Creative Commons Attribution license.)

Due to their unique properties, human mesenchymal stem/stromal cells (MSCs) possess tremendous potential in regenerative medicine, particularly in cell-based therapies where the multipotency and immunomodulatory characteristics of MSCs can be leveraged to address a variety of disease states. Although MSC-based cell therapeutics have emerged as one of the most promising medical treatments, the clinical translation is hampered by the variability of MSC-based cellular products caused by tissue source-specific differences and the lack of physiological cell culture approaches that closely mimic the human cellular microenvironment. In this study, a model for trilineage differentiation of primary adipose-, bone marrow-, and umbilical cord-derived MSCs into adipocytes, chondrocytes and osteoblasts was established and characterized. Differentiation was performed in spheroid culture, using hypoxic conditions and serum-free and antibiotics-free medium. This platform was characterized for spheroid diameter and trilineage differentiation capacity reflecting functionality of differentiated cells, as indicated by lineage-specific extracellular matrix (ECM) accumulation and expression of distinct secreted markers. The presented model shows spheroid growth during the course of differentiation and successfully supports trilineage differentiation for MSCs from almost all tissue sources except for osteogenesis of umbilical cord-derived MSCs. These findings indicate that this platform provides a suitable and favorable environment for trilineage differentiation of MSCs from various tissue sources. Therefore, it poses a promising model to generate highly relevant biological data urgently required for clinical translation and therefore might be used in the future to generate in vitro microtissues, building blocks for tissue engineering or as disease models., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest. The author(s) declared that they were an editorial board member of Frontiers, at the time of submission. This had no impact on the peer review process and the final decision., (Copyright © 2024 Moldaschl, Chariyev-Prinz, Toegel, Keck, Hiden, Egger and Kasper.)

Tumor-on-chips (ToCs) are useful platforms for studying the physiology of tumors and evaluating the efficacy and toxicity of anti-cancer drugs. However, the design and fabrication of a ToC system is not a trivial venture. We introduce a user-friendly, flexible, 3D-printed microfluidic device that can be used to culture cancer cells or cancer-derived spheroids embedded in hydrogels under well-controlled environments. The system consists of two lateral flow compartments (left and right sides), each with two inlets and two outlets to deliver cell culture media as continuous liquid streams. The central compartment was designed to host a hydrogel in which cells and microtissues can be confined and cultured. We performed tracer experiments with colored inks and 40 kDa fluorescein isothiocyanate dextran to characterize the transport/mixing performances of the system. We also cultured homotypic (MCF7) and heterotypic (MCF7-BJ) spheroids embedded in gelatin methacryloyl hydrogels to illustrate the use of this microfluidic device in sustaining long-term micro-tissue culture experiments. We further demonstrated the use of this platform in anticancer drug testing by continuous perfusion of doxorubicin, a commonly used anti-cancer drug for breast cancer. In these experiments, we evaluated drug transport, viability, glucose consumption, cell death (apoptosis), and cytotoxicity. In summary, we introduce a robust and friendly ToC system capable of recapitulating relevant aspects of the tumor microenvironment for the study of cancer physiology, anti-cancer drug transport, efficacy, and safety. We anticipate that this flexible 3D-printed microfluidic device may facilitate cancer research and the development and screening of strategies for personalized medicine., (© 2024 IOP Publishing Ltd.)

Human extracellular 6-O-endosulfatases Sulf-1 and Sulf-2 are the only enzymes that post-synthetically alter the 6-O sulfation of heparan sulfate proteoglycans (HSPG), which regulates interactions of HSPG with many proteins. Oncogenicity of Sulf-2 in different cancers has been documented, and we have shown that Sulf-2 is associated with poor survival outcomes in head and neck squamous cell carcinoma (HNSCC). Despite its importance, limited information is available on direct protein-protein interactions of the Sulf-2 protein in the tumor microenvironment. In this study, we used monoclonal antibody (mAb) affinity purification and mass spectrometry to identify galectin-3-binding protein (LG3BP) as a highly specific binding partner of Sulf-2 in the conditioned media of HNSCC cell lines. We validated their direct interaction in vitro using recombinant proteins and have shown that the chondroitin sulfate (CS) covalently bound to the Sulf-2 influences the binding to LG3BP. We confirmed the importance of the CS chain for the interaction by generating a mutant Sulf-2 protein that lacks the CS. Importantly, we have shown that the LG3BP inhibits Sulf-2 activity in vitro in a concentration-dependent manner. As a consequence, the addition of LG3BP to a spheroid cell culture inhibited the invasion of the HNSCC cells into Matrigel. Thus, Sulf-2 interaction with LG3BP may regulate the physiological activity of the Sulf-2 enzyme as well as its activity in the tumor microenvironment., Competing Interests: Conflict of interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 The Authors. Published by Elsevier Inc. All rights reserved.)

Drug efficacy determined in preclinical research is difficult to transfer to clinical practice. This is mainly due to the use of oversimplified models omitting the effect of the tumor microenvironment and the presence of various cell types participating in the formation of tumors in vivo. In this study, we used robust three-dimensional models including spheroids grown from colon cancer cell lines and organotypic cultures prepared from the colorectal carcinoma tissue to test novel therapeutic strategies. We developed a multi-modal approach combining brightfield and fluorescence microscopy for evaluating drug effects on organotypic cultures. Combined treatment with 5-fluorouracil and disulfiram/copper efficiently eliminated cancer cells in these 3D models. Moreover, disulfiram/copper down-regulated the expression of markers associated with 5-fluorouracil resistance, such as thymidylate synthase and CD133/CD44. Thus, we propose combined therapy of 5-fluorouracil and disulfiram/copper for further testing as a treatment for colorectal carcinoma. In addition, we show that organotypic cultures are suitable models for anti-cancer drug testing.

Cell culture systems have been widely used to address fundamental questions in biology without sacrificing animals. Three-dimensional (3D) organoids provide more information on in vivo conditions than traditional culture systems because multiple cell types in organoids interact with each other in 3D structures. Despite extensive research and advances in the organoid field, some important limitations remain and need further consideration. In this review, we summarize how organoids are generated from pluripotent stem cells and describe the recent technical progress that has made organoids more similar to in vivo tissues for the application of organoids to modeling cancer. Lastly, we briefly discuss some limitations that have been raised in this field.

Human 3D liver microtissues/spheroids are powerful in vitro models to study drug-induced liver injury (DILI) but the small number of cells per spheroid limits the models’ usefulness to study drug metabolism. In this work, we scale up the number of spheroids on both a plate and a standardized organ-chip platform by factor 100 using a basic method which requires only limited technical expertise. We successfully generated up to 100 spheroids using polymer-coated microwells in a 96-well plate (= liver-plate) or organ-chip (= liver-chip). Liver-chips display a comparable cellular CYP3A4 activity, viability, and biomarker expression as liver spheroids for at least one week, while liver-plate cultures display an overall reduced hepatic functionality. To prove its applicability to drug discovery and development, the liver-chip was used to test selected reference compounds. The test system could discriminate toxicity of the DILI-positive compound tolcapone from its less hepatotoxic structural analogue entacapone, using biochemical and morphological readouts. Following incubation with diclofenac, the liver-chips had an increased metabolite formation compared to standard spheroid cultures. In summary, we generated a human liver-chip model using a standardized organ-chip platform which combines up to 100 spheroids and can be used for the evaluation of both drug safety and metabolism.

Cell therapy is one of the new treatment methods in which mesenchymal stem/stromal cell (MSCs) transplantation is one of the cells widely used in this field. The results of MSCs application in the clinic prove their therapeutic efficacy. For this reason, many clinical trials have been designed based on the application of MSCs for various diseases, especially inflammatory disease and regenerative medicine. These cells perform their therapeutic functions through multiple mechanisms, including the differentiative potential, immunomodulatory properties, production of therapeutic exosomes, production of growth factors and cytokines, and anti-apoptotic effects. Exosomes are nanosized extracellular vesicles (EVs) that change target cell functions by transferring different cargos. The therapeutic ability of MSCs-derived exosomes has been demonstrated in many studies. However, some limitations, such as the low production of exosomes by cells and the need for large amounts of them and also their limited therapeutic ability, have encouraged researchers to find methods that increase exosomes' therapeutic potential. One of these methods is the spheroid culture of MSCs. Studies show that the three-dimensional culture (3DCC) of MSCs in the form of multicellular spheroids increases the therapeutic efficacy of these cells in laboratory and animal applications. In addition, the spheroid culture of MSCs leads to enhanced therapeutic properties of their exosomes and production rate. Due to the novelty of the field of using 3DCC MSCs-derived exosomes, examination of their properties and the results of their therapeutic application can increase our view of this field. This review discussed MSCs and their exosomes enhanced properties in spheroid culture.

Tumor spheroids play an increasingly important role in cancer research. Their ability to recapitulate crucial features of tumor biology that are lost in the classically used 2D models along with their relative simplicity and handiness have made them the most studied 3D tumor model. Their application as a theranostic tool or as a means to study tumor-host interaction is now well-established in various cancers. However, their use in the field of Renal Cell Carcinoma (RCC) remains very limited. The aim of this work is to present methods to implement a basic RCC spheroid model. These methods cover the steps from RCC tumor dissociation to spheroid infiltration by immune cells. We present a protocol for RCC dissociation using Liberase TM and introduce a culture medium containing Epithelial Growth Factor and Hydrocortisone allowing for faster growth of RCC primary cells. We show that the liquid overlay technique allows for the formation of spheroids from cell lines and from primary cultures. We present a method using morphological criteria to select a homogeneous spheroid population based on a Fiji macro. We then show that spheroids can be infiltrated by PBMCs after activation with OKT3 or IL-15. Finally, we provide an example of application by implementing an immune spheroid killing assay allowing observing increased spheroid destruction after treatment with PD-1 inhibitors. Thus the straightforward methods presented here allow for efficient spheroid formation for a simple RCC 3D model that can be standardized and infused with immune cells to study immunotherapies.

Breast cancer (BC) is a heterogenous disease classified into four molecular subtypes (Luminal A, Luminal B, HER2 and triple-negative (TNBC)) depending on the expression of the estrogen receptor (ER), the progesterone receptor (PR) and the human epidermal receptor 2 (HER2). The development of effective treatments for BC, especially TNBC, remains a challenge. Aminosteroid derivative RM-581 has previously shown an antiproliferative effect in multiple cancers in vitro and in vivo. In this study, we evaluated its effect in BC cell lines representative of BC molecular subtypes, including metastatic TNBC. We found that RM-581 has an antiproliferative effect on all BC molecular subtypes, especially on Luminal A and TNBC, in 2D and 3D cultures. The combination of RM-581 and trastuzumab or trastuzumab-emtansine enhanced the anticancer effect of each drug for HER2-positive BC cell lines, and the combination of RM-581 and taxanes (docetaxel or paclitaxel) improved the antiproliferative effect of RM-581 in TNBC and metastatic TNBC cell lines. We also confirmed that RM-581 is an endoplasmic reticulum (EnR)-stress aggravator by inducing an increase in EnR-stress-induced apoptosis markers such as BIP/GRP78 and CHOP and disrupting lipid homeostasis. This study demonstrates that RM-581 could be effective for the treatment of BC, especially TNBC.

In order to improve the conversion and transmission efficiency of the photoelectron, core–shell spheroid structure titanium dioxide/cadmium sulfide (TiO2/CdS) composites were synthesized as epoxy-based coating fillers using a simple hydrothermal method. The electrochemical performance of photocathodic protection for the epoxy-based composite coating was analyzed by coating it on the Q235 carbon steel surface. The results show that the epoxy-based composite coating possesses a significant photoelectrochemical property with a photocurrent density of 0.0421 A/cm2 and corrosion potential of −0.724 V. Importantly, the modified composite coating can extend absorption in the visible region and effectively separate photoelectron hole pairs to improve the photoelectrochemical performance synergistically, because CdS can be regarded as a sensitizer to be introduced into TiO2 to form a heterojunction system. The mechanism of photocathodic protection is attributed to the potential energy difference between Fermi energy and excitation level, which leads to the system obtaining higher electric field strength at the heterostructure interface, thus driving electrons directly into the surface of Q235 carbon steel (Q235 CS). Moreover, the photocathodic protection mechanism of the epoxy-based composite coating for Q235 CS is investigated in this paper.

Ovarian cancer (OC) is one of the most common gynecological cancers, with the worst prognosis and the highest mortality rate. Peritoneal dissemination (or carcinomatosis) accompanied by ascites formation is the most unfavorable factor in the progression and recurrence of OC. Tumor cells in ascites are present as either separate cells or, more often, as cell aggregates, i.e., spheroids which promote implantation on the surface of nearby organs and, at later stages, metastases to distant organs. Malignant ascites comprises a unique tumor microenvironment; this fact may be of relevance in the search for new prognostic and predictive factors that would make it possible to personalize the treatment of patients with OC. However, the precise mechanisms of spheroid formation and carcinomatosis are still under investigation. Here, we summarize data on ascites composition as well as the activity of fibroblasts and macrophages, the key stromal and immune components, in OC ascites. We describe current knowledge about the role of fibroblasts and macrophages in tumor spheroid formation, and discuss the specific functions of fibroblasts, macrophages and T cells in tumor peritoneal dissemination and implantation. [ABSTRACT FROM AUTHOR]

We studied the effect of 3D-culturing of cells in the form of cardiospheres on the expression of genes encoding vascular progenitor cell markers and angiogenesis regulators and on the production of proangiogenic factors. Cardiospheres were obtained by culturing mouse cardiac explants followed by self-assembly on poly-D-lysine. Gene expression was assessed by real-time PCR, and the production of proangiogenic factors was assessed by Microarray analysis of the cell secretome. It was found that cells in the cardiospheres in comparison with 2D-culture of cardiosphere-forming cells demonstrated increased expression of vascular progenitor cell markers (Pdgfrα, Kit, and Vegfr1) and angiogenesis regulatory factors (Vegf, Fgf2, and Angpt1), as well as an enhanced secretion of proangiogenic factors (ANGPT1, VEGF, CXCL16, and PIGF-2). Thus, culturing of cells in the form of cardiospheres can be considered as a basis for developing approaches to increasing their angiogenic activity and regenerative properties. [ABSTRACT FROM AUTHOR]

Stem cell transplantation is expected to be an effective treatment for intractable skin ulcers by promoting angiogenesis; however, it is challenging to quickly realize a sufficient bloodstream for the ulcers. For this treatment, sheet‐like materials with monolayer cells such as cell sheets have been investigated. However, they have a limitation of cell number that can be transplanted at one time due to the two‐dimensional, monolayer cell structure, and sufficient secretion of growth factors cannot be expected. In this regard, cellular aggregates, such as spheroids, can reproduce three‐dimensional cell‐cell interactions that cause biological functions of living tissues more representative than monolayer cells, which is important to achieving efficient secretion of growth factors. In this study, we focused on free‐standing porous polymer ultrathin films ("porous nanosheets") comprising poly(d,l‐lactic acid) (PDLLA) and succeeded in developing a spheroid‐covered nanosheet, on which more than 1000 spheroids from adipose‐tissue derived stem cells (ASCs) were loaded. The porous structure with an average pore diameter of 4 μm allowed for facile filtration and carrying spheroids on the nanosheet, as well as sufficient oxygen and nutrients inflow to the cells. The spheroid‐covered nanosheet achieved homogeneous transference of spheroids to a whole skin defect in diabetic model mice. Given the continuous release of vascular endothelial growth factor (VEGF) from the spheroids, the transplanted spheroids promoted healing with more accelerated angiogenesis than a nanosheet with a monolayer of cells. The spheroid‐covered nanosheet may be a new regenerative material for promoting intractable skin ulcer healing. [ABSTRACT FROM AUTHOR]

Background: Muscle denervation from trauma and motor neuron disease causes disabling morbidities. A limiting step in functional recovery is the regeneration of neuromuscular junctions (NMJs) for reinnervation. Stem cells have the potential to promote these regenerative processes, but current approaches have limited success, and the optimal types of stem cells remain to be determined. Neural crest stem cells (NCSCs), as the developmental precursors of the peripheral nervous system, are uniquely advantageous, but the role of NCSCs in neuromuscular regeneration is not clear. Furthermore, a cell delivery approach that can maintain NCSC survival upon transplantation is critical. Methods: We established a streamlined protocol to derive, isolate, and characterize functional p75+ NCSCs from human iPSCs without genome integration of reprogramming factors. To enhance survival rate upon delivery in vivo, NCSCs were centrifuged in microwell plates to form spheroids of desirable size by controlling suspension cell density. Human bone marrow mesenchymal stem cells (MSCs) were also studied for comparison. NCSC or MSC spheroids were injected into the gastrocnemius muscle with denervation injury, and the effects on NMJ formation and functional recovery were investigated. The spheroids were also co-cultured with engineered neuromuscular tissue to assess effects on NMJ formation in vitro. Results: NCSCs cultured in spheroids displayed enhanced secretion of soluble factors involved in neuromuscular regeneration. Intramuscular transplantation of spheroids enabled long-term survival and retention of NCSCs, in contrast to the transplantation of single-cell suspensions. Furthermore, NCSC spheroids significantly improved functional recovery after four weeks as shown by gait analysis, electrophysiology, and the rate of NMJ innervation. MSC spheroids, on the other hand, had insignificant effect. In vitro co-culture of NCSC or MSC spheroids with engineered myotubes and motor neurons further evidenced improved innervated NMJ formation with NCSC spheroids. Conclusions: We demonstrate that stem cell type is critical for neuromuscular regeneration and that NCSCs have a distinct advantage and therapeutic potential to promote reinnervation following peripheral nerve injury. Biophysical effects of spheroidal culture, in particular, enable long-term NCSC survival following in vivo delivery. Furthermore, synthetic neuromuscular tissue, or "tissues-on-a-chip," may offer a platform to evaluate stem cells for neuromuscular regeneration. [ABSTRACT FROM AUTHOR]

In this paper we present an efficient Matlab computation of a 3-D electromagnetic scattering problem, in which a plane wave impinges with a generic inclination onto a conducting ellipsoid of revolution. This solid is obtained by the rotation of an ellipse around one of its axes, which is also known as a spheroid. We have developed a fast and ad hoc code to solve the electromagnetic scattering problem, using spheroidal vector wave functions, which are special functions used to describe physical problems in which a prolate or oblate spheroidal reference system is considered. Numerical results are presented, both for TE and TM polarization of the incident wave, and are validated by a comparison with results obtained by a commercial electromagnetic simulator. [ABSTRACT FROM AUTHOR]

Adipose-derived stem cells (ADSCs), due to their regenerative ability, have beneficial effects on bone and cartilage defects. In addition, spheroid formation of ADSCs obtained using three-dimensional (3D) culture accelerates the regenerative ability of ADSCs. The study investigated the regenerative effect of 3D-cultured small size ADSC spheroids without a scaffold in rats with defects in the critical-sized calvarial bone. ADSC-single cells, ADSC-spheroids, or PBS (as control) were implanted in rats, and radiological and histological assessment of bone regeneration was performed. Bone defects were significantly regenerated in the ADSC-spheroid group compared to that in the control group. ADSC-spheroids also showed the most significant bone regeneration in histological assessment. Immunohistochemistry assessment showed that ADSC-spheroids could survive 12 weeks after cell implantation. In vitro , cell apoptosis in ADSC-spheroids was significantly suppressed compared to that in ADSC-single cells. In addition, gene expression related to bone morphogenesis, angiogenesis, and stemness in ADSC-spheroids was elevated. The scaffold-free 3D-cultured small ADSC-spheroids survived in in vitro and in vivo conditions and promoted bone regeneration. Therefore, injectable small size ADSC-spheroids are a novel and less-invasive therapeutic option for treating bone defects. • Successful use of ADSC spheroids in bone regeneration without a scaffold. • Paracrine secretions in spheroids enhance bone regeneration. • MicoCell is a novel multi-cavity cell culture platform to grow small spheroids. [ABSTRACT FROM AUTHOR]

Simple Summary: Ultrasound waves can be applied for diagnostic and therapeutic purposes. Focused ultrasound is approved for tissue ablation, e.g., in the treatment of uterine fibroids or essential tremors. Besides the non-invasive image-guided surgical intervention at temperatures above 55 °C, FUS is investigated in other fields like blood-brain barrier opening, hyperthermia, and neuromodulation. FUS offers potential as an adjuvant therapy in cancer treatment. Therefore, analysis of FUS effects on cancer cells is necessary. We performed studies on two human cancer cell line spheroids using a newly developed high-throughput in vitro FUS applicator with 32 individual transducers. This study aimed to perform basic experiments with a new in vitro FUS device on a 3D tumour model to acquire insight into the effects of FUS at the cellular level. These experiments may contribute to a better understanding and predictions of cancer treatment efficacy. Focused ultrasound (FUS) is a non-invasive technique producing a variety of biological effects by either thermal or mechanical mechanisms of ultrasound interaction with the targeted tissue. FUS could bring benefits, e.g., tumour sensitisation, immune stimulation, and targeted drug delivery, but investigation of FUS effects at the cellular level is still missing. New techniques are commonly tested in vitro on two-dimensional (2D) monolayer cancer cell culture models. The 3D tumour model—spheroid—is mainly utilised to mimic solid tumours from an architectural standpoint. It is a promising method to simulate the characteristics of tumours in vitro and their various responses to therapeutic alternatives. This study aimed to evaluate the effects of FUS on human prostate and glioblastoma cancer tumour spheroids in vitro. The experimental follow-up enclosed the measurements of spheroid integrity and growth kinetics, DNA damage, and cellular metabolic activity by measuring intracellular ATP content in the spheroids. Our results showed that pulsed FUS treatment induced molecular effects in 3D tumour models. With the disruption of the spheroid integrity, we observed an increase in DNA double-strand breaks, leading to damage in the cancer cells depending on the cancer cell type. [ABSTRACT FROM AUTHOR]

Three-dimensional cell culture techniques mimic the in vivo cell environment more adequately than flat surfaces. Spheroids are multicellular aggregates and we aimed to produce scaffold-free spheroids of myogenic origin, called myospheres, using a mid-scale incubator and bioreactor hybrid. For the first time, we obtained spheroids from primary porcine muscle cells (PMCs) with this technology and compared their morphology and growth parameters, marker expression, and myogenic potential to C2C12-derived spheroids. Both cell types were able to form round-shaped spheroids in the bioreactor already after 24 h. The mean diameter of the C2C12 spheroids (44.6 µm) was larger than that of the PMCs (32.7 µm), and the maximum diameter exceeded 1 mm. C2C12 cells formed less aggregates than PMCs with a higher packing density (cell nuclei/mm2). After dissociation from the spheroids, C2C12 cells and PMCs started to proliferate again and were able to differentiate into the myogenic lineage, as shown by myotube formation and the expression of F-Actin, Desmin, MyoG, and Myosin. For C2C12, multinucleated syncytia and Myosin expression were observed in spheroids, pointing to accelerated myogenic differentiation. In conclusion, the mid-scale incubator and bioreactor system is suitable for spheroid formation and cultivation from primary muscle cells while preserving their myogenic potential. [ABSTRACT FROM AUTHOR]

Alzheimer's disease (AD) is an incurable neurodegenerative disorder and the leading cause of death among older individuals. Available treatment strategies only temporarily mitigate symptoms without modifying disease progression. Recent studies revealed the multifaceted neurobiology of AD and shifted the target of drug development. Established animal models of AD are mostly tailored to yield a subset of disease phenotypes, which do not recapitulate the complexity of sporadic late-onset AD, the most common form of the disease. The use of human induced pluripotent stem cells (HiPSCs) offers unique opportunities to fill these gaps. Emerging technology allows the development of disease models that recapitulate a brain-like microenvironment using patient-derived cells. These models retain the individual's unraveled genetic background, yielding clinically relevant disease phenotypes and enabling cost-effective, high-throughput studies for drug discovery. Here, we review the development of various HiPSC-based models to study AD mechanisms and their application in drug discovery. [ABSTRACT FROM AUTHOR]

Spheroids have become an essential tool in preclinical cancer research. The uniformity of spheroids is a critical parameter in drug test results. Spheroids form by self-assembly of cells. Hence, the control of homogeneity of spheroids in terms of size, shape, and density is challenging. We developed surface-optimized polydimethylsiloxane (PDMS) biochip platforms for uniform spheroid formation on-chip. These biochips were surface modified with 10% bovine serum albumin (BSA) to effectively suppress cell adhesion on the PDMS surface. These surface-optimized platforms facilitate cell self-aggregations to produce homogenous non-scaffold-based spheroids. We produced uniform spheroids on these biochips using six different established human cell lines and a co-culture model. Here, we observe that the concentration of the BSA is important in blocking cell adhesion to the PDMS surfaces. Biochips treated with 3% BSA demonstrated cell repellent properties similar to the bare PDMS surfaces. This work highlights the importance of surface modification on spheroid production on PDMS-based microfluidic devices. [ABSTRACT FROM AUTHOR]

The development of novel cancer therapeutic strategies has garnered increasing interest in cancer research. Among the therapeutic choices, chemosensitizers have shown exciting prospects. Peptides are an attractive alternative among the molecules that may be used as chemosensitizers. We rationally designed a new-to-nature peptide, nurP28, derived from the 22-kDa α-zein protein sequence (entry Q00919_MAIZE). The resultant sequence of the nurP28 peptide after the addition of arginine residues was LALLALLRLRRRATTAFIIP, and we added acetyl and amide groups at the N- and C-terminus, respectively, for capping. We evaluated the cytotoxicity of the nurP28 peptide alone and in combination with docetaxel in fibroblast monolayers and breast cancer monolayers and spheroids. Our results indicated that nurP28 is not cytotoxic to human fibroblasts or cancer cells. Nevertheless, when combined with 1 µM docetaxel, 3 ng/mL nurP28 induced equivalent (in MCF7 monolayers) and higher (in MCF7 spheroids) cytotoxic effects than 10-fold higher doses of docetaxel alone. These findings suggest that nurP28 may act as a chemosensitizer in breast cancer treatment. This study describes the enhancing "anti-cancer" effects of nurP28 in breast cancer 2D and 3D cultures treated with docetaxel. Further studies should explore the mechanisms underlying these effects and assess the clinical potential of our findings using animal models. [ABSTRACT FROM AUTHOR]

Background: In vitro three-dimensional (3D) hepatic spheroid culture has shown great promise in toxicity testing because it better mimics the cell–cell and cell–matrix interactions found in in vivo conditions than that of the traditional two-dimensional (2D) culture. Despite embedding HepaRG spheroids with collagen type I (collagen I) extracellular matrix (ECM) revealed a much better differentiation capability, almost all the collagen utilized in in vitro hepatocytes cultures is animal-derived collagen that may limit its use in human toxicity testing. Method: Here, a preliminary investigation of HepaRG cells cultured in different dimensionalities and with the addition of ECM was performed. Comparisons of conventional 2D culture with 3D spheroid culture were performed based on their functional or structural differences over 7 days. Rat tail collagen (rtCollagen) I and recombinant human collagen (rhCollagen) I were investigated for their ability in promoting HepaRG spheroid differentiation. Results: An immunofluorescence analysis of the hepatocyte-specific functional protein albumin suggested that HepaRG spheroids demonstrated better hepatic function than spheroids from 2D culture, and the function of HepaRG spheroids improved in a time-dependent manner. The fluorescence intensities per unit area of spheroids formed by 1000 cells on days 7 and 10 were 25.41 and 45.38, respectively, whereas almost undetectable fluorescence was obtained with 2D cells. In addition, the embedding of HepaRG spheroids into rtCollagen and rhCollagen I showed that HepaRG differentiation can be accelerated relative to the differentiation of spheroids grown in suspension, demonstrating the great promise of HepaRG spheroids. Conclusions: The culture conditions established in this study provide a potentially novel alternative for promoting the differentiation of HepaRG spheroids into mature hepatocytes through a collagen-embedded in vitro liver spheroid model. This culture method is envisioned to provide insights for future drug toxicology. [ABSTRACT FROM AUTHOR]

Purpose: To report second-look arthroscopic assessment after all-arthroscopic autologous chondrocyte implantation (ACI) for articular cartilage defects at the patella. Methods: A second-look arthroscopy after all-arthroscopic ACI using chondrospheres® (ACT3D) was performed in 30 patients with 30 full-thickness retropatellar cartilage defects. The mean time from ACI to second-look arthroscopy was 14.9 ± 16.3 (6–71) months. The quality of cartilage regeneration was evaluated by the International Cartilage-Repair Score (ICRS)—Cartilage Repair Assessment (CRA). Results: Eleven lesions (36.7%) were classified as CRA grade I (normal) and 19 lesions (63.3%) as grade II (nearly normal). Concerning the degree of defect repair, 25 lesions (83.3%) were repaired up to the height of the surrounding articular retropatellar cartilage. Five lesions (16.7%) showed 75% repair of defect depth. The border zone was completely integrated into the surrounding articular cartilage shoulder in 28 lesions (93.3%) and demarcated within 1 mm in 2 lesions (6.7%). Macroscopically and by probing, 12 lesions (40%) had intact smooth surface, 17 lesions (56.7%) had fibrillated surface and 1 lesion (3.3%) had small, scattered fissures. A negative correlation was found between the overall repair assessment score and the defect size (r2 = − 0.430, p = 0.046) and between integration into border zone and defect size (r2 = − 0.340, p = 0.045). A positive correlation was found between macroscopic appearance and age (r2 = + 0.384, p = 0.036). Conclusions: All-arthroscopic ACI using chondrospheres® (ACT3D) for full-thickness retropatellar articular cartilage defects proved to be reproducible and reliable. The advantage of the procedure is that it is minimal invasive. Arthroscopic second-look demonstrated a high grade of normal or nearly normal cartilage regeneration. Although statistically significant differences were not observed, larger defect size and younger age may compromise the result of overall repair. Level of evidence: III. [ABSTRACT FROM AUTHOR]

Mesenchymal stem cells such as human adipose tissue‐derived stem cells (hADSCs) have been used as a representative therapeutic agent for tissue regeneration because of their high proliferation and paracrine factor‐secreting abilities. However, certain points regarding conventional ADSC delivery systems, such as low cell density, secreted cytokine levels, and cell viability, still need to be addressed for treating severe wounds. In this study, we developed a three‐dimensional (3D) cavity‐structured stem cell‐laden system for overdense delivery of cells into severe wound sites. Our system includes a hydrophobic surface and cavities that can enhance the efficiency of cell delivery to the wound site. In particular, the cavities in the system facilitate hADSC spheroid formation, increasing therapeutic growth factor expression compared with 2D cultured cells. Our hADSC spheroid‐loaded patch exhibited remarkably improved cell localization at the wound site and dramatic therapeutic efficacy compared to the conventional cell injection method. Taken together, the hADSC spheroid delivery system focused on cell delivery, and stem cell homing effect at the wound site showed a significantly enhanced wound healing effect. By overcoming the limitations of conventional cell delivery methods, our overdense cell delivery system can contribute to biomedical and clinical applications. [ABSTRACT FROM AUTHOR]