Search

Three dimensional models of cell culture enables researchers to recreate aspects of tumour biology not replicated by traditional two dimensional techniques. Here we describe a protocol to enable automated high throughput phenotypic profiling across panels of patient derived glioma stem cell spheroid models. We demonstrate the use of both live/dead cell end-points and monitor the dynamic changes in the cell cycle using cell lines expressing the FUCCI cell cycle reporter. Together, these assays provide additional insight into the mechanism of action of compound treatments over traditional cell viability assay endpoints.

Three-dimensional (3D) stem cell culture systems have attracted considerable attention as a way to better mimic the complex interactions between individual cells and the extracellular matrix (ECM) that occur in vivo. Moreover, 3D cell culture systems have unique properties that help guide specific functions, growth, and processes of stem cells (e.g., embryogenesis, morphogenesis, and organogenesis). Thus, 3D stem cell culture systems that mimic in vivo environments enable basic research about various tissues and organs. In this review, we focus on the advanced therapeutic applications of stem cell-based 3D culture systems generated using different engineering techniques. Specifically, we summarize the historical advancements of 3D cell culture systems and discuss the therapeutic applications of stem cell-based spheroids and organoids, including engineering techniques for tissue repair and regeneration.

Multicellular tumor spheroids (MTSs) are cells grown in aggregates to form a 3D tumor model. While 20 cell culture-cells grown in a monolayer-is useful for cancer research and drug screening, the response is often variable from what would occur in vivo. 3D cell culture mimics the tumor micro-environment and contains other similarities to in vivo tumors, such as necrotic and hypoxic regions, allowing for a more realistic prediction of treatment response. In this study, the protocol for generating MTSs from multiple triple-negative breast cancer (TNBC) cell lines was developed to use as a drug screening tool for potential treatments. For each cell line, an MTS model was developed and characterized to determine the cell number required to achieve spheroids with a diameter of -400 microns after 72 hours of growth. All spheroids were generated in low-adhesion cell culture plates. Developed spheroids were then tested in drug screening assays, and results were compared to previous 2D results. Data collection included final spheroid diameter, cell density, and cell viability count to assess the effects of administered treatment. This model will be used in future studies to better understand the impacts ofdrug treatments in a inore translational model. [ABSTRACT FROM AUTHOR]

Oxygen consumption has been used to evaluate various cellular activities. In addition, three-dimensional (3D) spheroids have been broadly exploited as advanced in vitro cell models for various biomedical studies due to their capability of mimicking 3D in vivo microenvironments and cell arrangements. However, monitoring the oxygen consumption of live 3D spheroids poses challenges because existing invasive methods cause structural and cell damage. In contrast, optical methods using fluorescence labeling and microscopy are non-invasive, but they suffer from technical limitations like high cost, tedious procedures, and poor signal-to-noise ratios. To address these challenges, we developed a microfluidic platform for uniform-sized spheroid formation, handling, and culture. The platform is further integrated with widefield frequency domain fluorescence lifetime imaging microscopy (FD-FLIM) to efficiently characterize the lifetime of an oxygen-sensitive dye filling the platform for oxygen consumption characterization. In the experiments, osteosarcoma (MG-63) cells are exploited as the spheroid model and for the oxygen consumption analysis. The results demonstrate the functionality of the developed approach and show the accurate characterization of the oxygen consumption of the spheroids in response to drug treatments. The developed approach possesses great potential to advance spheroid metabolism studies with single-spheroid resolution and high sensitivity.

The clinical application of mesenchymal stem cells (MSCs) is attracting attention due to their excellent safety, convenient acquisition, multipotency, and trophic activity. The clinical effectiveness of transplanted MSCs is well-known in regenerative and immunomodulatory medicine, but there is a demand for their improved viability and regenerative function after transplantation. In this study, we isolated MSCs from adipose tissue from three human donors and generated uniformly sized MSC spheroids (∼100 µm in diameter) called microblocks (MiBs) for dermal reconstitution. The viability and MSC marker expression of MSCs in MiBs were similar to those of monolayer MSCs. Compared with monolayer MSCs, MiBs produced more extracellular matrix (ECM) components, including type I collagen, fibronectin, and hyaluronic acid, and growth factors such as vascular endothelial growth factor and hepatocyte growth factor. Subcutaneously injected MiBs showed skin volume retaining capacity in mice. These results indicate that MiBs could be applied as regenerative medicine for skin conditions such as atrophic scar by having high ECM and bioactive factor expression.

For ethical, economical, and scientific reasons, animal experimentation, used to evaluate the potential neurotoxicity of chemicals before their release in the market, needs to be replaced by new approach methodologies. To illustrate the use of new approach methodologies, the human induced pluripotent stem cell-derived 3D model BrainSpheres was acutely (48 h) or repeatedly (7 days) exposed to amiodarone (0.625–15 µM), a lipophilic antiarrhythmic drug reported to have deleterious effects on the nervous system. Neurotoxicity was assessed using transcriptomics, the immunohistochemistry of cell type-specific markers, and real-time reverse transcription–polymerase chain reaction for various genes involved in the lipid metabolism. By integrating distribution kinetics modeling with neurotoxicity readouts, we show that the observed time- and concentration-dependent increase in the neurotoxic effects of amiodarone is driven by the cellular accumulation of amiodarone after repeated dosing. The development of a compartmental in vitro distribution kinetics model allowed us to predict the change in cell-associated concentrations in BrainSpheres with time and for different exposure scenarios. The results suggest that human cells are intrinsically more sensitive to amiodarone than rodent cells. Amiodarone-induced regulation of lipid metabolism genes was observed in brain cells for the first time. Astrocytes appeared to be the most sensitive human brain cell type in vitro. In conclusion, assessing readouts at different molecular levels after the repeat dosing of human induced pluripotent stem cell-derived BrainSpheres in combination with the compartmental modeling of in vitro kinetics provides a mechanistic means to assess neurotoxicity pathways and refine chemical safety assessment for humans.

Spheroids are three-dimensional cell aggregates that mimic fundamental aspects of the native tissue microenvironment better than single cells, making them a promising platform for the study of tissue development and therapeutics. Spheroids have been investigated for decades as models in cancer research, yet we have only just scratched the surface of their potential clinical utility in cell-based therapies. Like many cells, spheroids commonly exhibit a loss of key attributes upon implantation, motivating the need for strategies to regulate their function in situ. Biomaterials offer numerous opportunities to preserve spheroid function and guide spheroid behavior by tailoring the local microenvironment.

Colorectal cancer (CRC) is a leading cause of death worldwide. Improved preclinical tumor models are needed to make treatment screening clinically relevant and address disease mortality. Advancements in 3D cell culture have enabled a greater recapitulation of the architecture and heterogeneity of the tumor microenvironment (TME). This has enhanced their pathophysiological relevance and enabled more accurate predictions of tumor progression and drug response in patients. An increasing number of 3D CRC spheroid models include cell populations such as cancer-associated fibroblasts (CAFs), endothelial cells (ECs), immune cells, and gut bacteria to better mimic the in vivo regulation of signaling pathways. Furthermore, cell heterogeneity within the 3D spheroid models enables the identification of new therapeutic targets to develop alternative treatments and test TME-target therapies. In this mini review, we present the advances in mimicking tumor heterogeneity in 3D CRC spheroid models by incorporating CAFs, ECs, immune cells, and gut bacteria. We introduce how, in these models, the diverse cells influence chemoresistance and tumor progression of the CRC spheroids. We also highlight important parameters evaluated during drug screening in the CRC heterocellular spheroids.

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. [ABSTRACT FROM AUTHOR]

Mitochondria are critical organelles responsible for the maintenance of cellular energy homeostasis. Thus, their dysfunction can have severe consequences in cells responsible for energy-intensive metabolic function, such as hepatocytes. Extensive research over the last decades have identified compromised mitochondrial function as a central feature in the pathophysiology of liver injury induced by an acetaminophen (APAP) overdose, the most common cause of acute liver failure in the United States. While hepatocyte mitochondrial oxidative and nitrosative stress coupled with induction of the mitochondrial permeability transition are well recognized after an APAP overdose, recent studies have revealed additional details about the organelle's role in APAP pathophysiology. This concise review highlights these new advances, which establish the central role of the mitochondria in APAP pathophysiology, and places them in the context of earlier information in the literature. Adaptive alterations in mitochondrial morphology as well as the role of cellular iron in mitochondrial dysfunction and the organelle's importance in liver recovery after APAP-induced injury will be discussed. [ABSTRACT FROM AUTHOR]

Induced pluripotent stem cells (iPSCs) may develop into any form of cell and are being intensively investigated. The influence on iPSCs of nanostructures generated using two-dimensional colloidal arrays was examined in this study. Colloidal arrays were formed using the following procedure. First, core–shell colloids were adsorbed onto a glass substrate using a layer-by-layer method. Second, the colloids were immobilized via thermal fusion. Third, the surface of the colloids was modified by plasma treatment. By adjusting the number density of colloids, cultured iPSCs were easily detached from the substrate without manual cell scraping. In addition to planar culture, cell aggregation of iPSCs attached to the substrate was achieved by combining hydrophilic surface patterning on the colloidal array. Multilayered cell aggregates with approximately four layers were able be cultured. These findings imply that colloidal arrays might be an effective tool for controlling the strength of cell adhesion. [ABSTRACT FROM AUTHOR]

Mercury (Hg) cytotoxicity, which is largely mediated through oxidative stress (OS), can be relieved with antioxidants. Thus, we aimed to study the effects of Hg alone or in combination with 5 nM N-Acetyl-L-cysteine (NAC) on the primary endometrial cells' viability and function. Primary human endometrial epithelial cells (hEnEC) and stromal cells (hEnSC) were isolated from 44 endometrial biopsies obtained from healthy donors. The viability of treated endometrial and JEG-3 trophoblast cells was evaluated via tetrazolium salt metabolism. Cell death and DNA integrity were quantified following annexin V and TUNEL staining, while the reactive oxygen species (ROS) levels were quantified following DCFDA staining. Decidualization was assessed through secreted prolactin and the insulin-like growth factor-binding protein 1 (IGFBP1) in cultured media. JEG-3 spheroids were co-cultured with the hEnEC and decidual hEnSC to assess trophoblast adhesion and outgrowth on the decidual stroma, respectively. Hg compromised cell viability and amplified ROS production in trophoblast and endometrial cells and exacerbated cell death and DNA damage in trophoblast cells, impairing trophoblast adhesion and outgrowth. NAC supplementation significantly restored cell viability, trophoblast adhesion, and outgrowth. As these effects were accompanied by the significant decline in ROS production, our findings originally describe how implantation-related endometrial cell functions are restored in Hg-treated primary human endometrial co-cultures by antioxidant supplementation. [ABSTRACT FROM AUTHOR]

Hepatic stellate cells (HSCs) play an important role in liver fibrosis; however, owing to the heterogeneity and limited supply of primary HSCs, the development of in vitro liver fibrosis models has been impeded. In this study, we established and characterized a novel human HSC line (LSC‐1), and applied it to various types of three‐dimensional (3D) co‐culture systems with differentiated HepaRG cells. Furthermore, we compared LSC‐1 with a commercially available HSC line on conventional monolayer culture. LSC‐1 exhibited an overall upregulation of the expression of fibrogenic genes along with increased levels of matrix and adhesion proteins, suggesting a myofibroblast‐like or transdifferentiated state. However, activated states reverted to a quiescent‐like phenotype when cultured in different 3D culture formats with a relatively soft microenvironment. Additionally, LSC‐1 exerted an overall positive effect on co‐cultured differentiated HepaRG, which significantly increased hepatic functionality upon long‐term cultivation compared with that achieved with other HSC line. In 3D spheroid culture, LSC‐1 exhibited enhanced responsiveness to transforming growth factor beta 1 exposure that is caused by a different matrix‐related protein expression mechanism. Therefore, the LSC‐1 line developed in this study provides a reliable candidate model that can be used to address unmet needs, such as development of antifibrotic therapies. [ABSTRACT FROM AUTHOR]

Sarcomas are rare malignancies, the number of reports is limited, and this rarity makes further research difficult even though liposarcoma is one of major sarcomas. 2D cell culture remains an important role in establishing basic tumor biology research, but its various shortcomings and limitations are still of concern, and it is now well-accepted that the behavior of 3D-cultured cells is more reflective of in vivo cellular responses compared to 2D models. This study aimed to establish 3D cell culture of liposarcomas using two different methods: scaffold-based (Matrigel extracellular matrix [ECM] scaffold method) and scaffold-free (Ultra-low attachment [ULA] plate). Lipo246, Lipo224 and Lipo863 cell lines were cultured, and distinctive differences in structures were observed in Matrigel 3D model: Lipo224 and Lipo863 formed spheroids, whereas Lipo246 grew radially without forming spheres. In ULA plate approaches, all cell lines formed spheroids, but Lipo224 and Lipo863 spheroids showed bigger size and looser aggregation than Lipo246. Formalin fixed, paraffin embedded (FFPE) blocks were obtained from all 3D models, confirming the spheroid structures. The expression of MDM2, Ki-67 positivity and MDM2 amplification were confirmed by IHC and DNAscope™, respectively. Protein and DNA were extracted from all samples and MDM2 upregulation was confirmed by western blot and qPCR analysis. After treatment with MDM2 inhibitor SAR405838, DDLPS spheroids demonstrated different sensitivity patterns from 2D models. Taken together, we believed that 3D models would have a possibility to provide us a new predictability of efficacy and toxicity, and considered as one important process in in vitro pre-clinical phase prior to moving forward to clinical trials. [ABSTRACT FROM AUTHOR]

Multicellular tumor spheroids are rapidly emerging as an improved in vitro model with respect to more traditional 2D culturing. Microwell culturing is a simple and accessible method for generating a large number of uniformly sized spheroids, but commercially available systems often do not enable researchers to perform complete culturing and analysis pipelines and the mechanical properties of their culture environment are not commonly matching those of the target tissue. We herein report a simple method to obtain custom-designed self-built microwell arrays made of polydimethylsiloxane or agarose for uniform 3D cell structure generation. Such materials can provide an environment of tunable mechanical flexibility. We developed protocols to culture a variety of cancer and non-cancer cell lines in such devices and to perform molecular and imaging characterizations of the spheroid growth, viability, and response to pharmacological treatments. Hundreds of tumor spheroids grow (in scaffolded or scaffold-free conditions) at homogeneous rates and can be harvested at will. Microscopy imaging can be performed in situ during or at the end of the culture. Fluorescence (confocal) microscopy can be performed after in situ staining while retaining the geographic arrangement of spheroids in the plate wells. This platform can enable statistically robust investigations on cancer biology and screening of drug treatments. [ABSTRACT FROM AUTHOR]

Background: Adipose tissue-derived stromal vascular fraction (SVF) harbors multipotent cells with potential therapeutic relevance. We developed a method to form adipose spheroids (AS) from the SVF with complex organoid structure and enhanced leptin secretion upon insulin stimulation. Methods: SVF was generated from the interscapular brown adipose tissue of newborn mice. Immunophenotype and stemness of cultured SVF were determined by flow cytometry and in vitro differentiation, respectively. Spheroids were generated in hanging drops and non-adherent plates and compared by morphometric methods. The adipogenic potential was compared between preadipocyte monolayers and spheroids. Extracellular leptin was quantified by immunoassay. Lipolysis was stimulated with isoprenaline and quantified by colorimetric methods. AS viability and ultrastructure were determined by confocal and transmission electron microscopy analyses. Results: Cultured SVF contained Sca1 + CD29 + CD44 + CD11b- CD45- CD90- cells with adipogenic and chondrogenic but no osteogenic potential. Culture on non-adherent plates yielded the highest quantity and biggest size of spheroids. Differentiation of AS for 15 days in a culture medium supplemented with insulin and rosiglitazone resulted in greater Pparg, Plin1, and Lep expression compared to differentiated adipocytes monolayers. AS were viable and maintained leptin secretion even in the absence of adipogenic stimulation. Glycerol release after isoprenaline stimulation was higher in AS compared to adipocytes in monolayers. AS were composed of outer layers of unilocular mature adipocytes and an inner structure composed of preadipocytes, immature adipocytes and an abundant loose extracellular matrix. Conclusion: Newborn mice adipose SVF can be efficiently differentiated into leptin-secreting AS. Prolonged stimulation with insulin and rosiglitazone allows the formation of structurally complex adipose organoids able to respond to adrenergic lipolytic stimulation. [ABSTRACT FROM AUTHOR]

Current pre-clinical models of cancer fail to recapitulate the cancer cell behavior in primary tumors primarily because of the lack of a deeper understanding of the effects that the microenvironment has on cancer cell phenotype. Transcriptomic profiling of 4T1 murine mammary carcinoma cells from 2D and 3D cultures, subcutaneous or orthotopic allografts (from immunocompetent or immunodeficient mice), as well as ex vivo tumoroids, revealed differences in molecular signatures including altered expression of genes involved in cell cycle progression, cell signaling and extracellular matrix remodeling. The 3D culture platforms had more in vivo-like transcriptional profiles than 2D cultures. In vivo tumors had more cells undergoing epithelial-to-mesenchymal transition (EMT) while in vitro cultures had cells residing primarily in an epithelial or mesenchymal state. Ex vivo tumoroids incorporated aspects of in vivo and in vitro culturing, retaining higher abundance of cells undergoing EMT while shifting cancer cell fate towards a more mesenchymal state. Cellular heterogeneity surveyed by scRNA-seq revealed that ex vivo tumoroids, while rapidly expanding cancer and fibroblast populations, lose a significant proportion of immune components. This study emphasizes the need to improve in vitro culture systems and preserve syngeneic-like tumor composition by maintaining similar EMT heterogeneity as well as inclusion of stromal subpopulations.

The expression of pluripotency factors, and their associations with clinicopathological parameters and drug response have been described in various cancers, including gastric cancer. This study investigated the association of pluripotency factor expression with the clinicopathological characteristics of gastric cancer patients, as well as changes in the expression of these factors upon the stem cell-enriching spheroid culture of gastric cancer cells, regulation of sphere-forming capacity, and response to cisplatin and TRAIL treatments by Nanog and KLF4. Nanog expression was significantly associated with the emergence of a new tumor and a worse prognosis in gastric cancer patients. The expression of the pluripotency factors varied among six gastric cancer cells. KLF4 and Nanog were expressed high in SNU-601, whereas SOX2 was expressed high in SNU-484. The expression of KLF4 and SOX2 was increased upon the spheroid culture of SNU-601 (KLF4/Nanog-high) and SNU-638 (KLF4/Nanog-low). The spheroid culture of them enhanced TRAIL-induced viability reduction, which was accompanied by the upregulation of death receptors, DR4 and DR5. Knockdown and overexpression of Nanog in SNU-601 and SNU-638, respectively, did not affect spheroid-forming capacity, however, its expression was inversely correlated with DR4/DR5 expression and TRAIL sensitivity. In contrast, KLF4 overexpression in SNU-638 increased spheroid formation, susceptibility to cisplatin and TRAIL treatments, and DR4/DR5 expression, while the opposite was found in KLF4-silenced SNU-601. KLF4 is supposed to play a critical role in DR4/DR5 expression and responses to TRAIL and cisplatin, whereas Nanog is only implicated in the former events only. Direct regulation of death receptor expression and TRAIL response by KLF4 and Nanog have not been well documented previously, and the regulatory mechanism behind the process remains to be elucidated.

Emerging bioengineering approaches such as three-dimensional (3D) cell culture methods have opened new avenues for scaffolds combined with various therapeutic agents in bone tissue regeneration. In this regard, bioceramic nanocomposite compounds have been prominently studied in bone and dental tissue engineering, due to their potential for diseased bone regeneration. We synthesized in situ bioceramic nanocomposite using a combination of sol–gel and mechanical activation that the nanocomposite contained Akermanite as a major phase, and Monticellite as a minor phase. After physicochemical characterization (crystallite size of Ak and Mon were 58 and 48 nm respectively), cell viability and pertinent biological studies of the nanocomposite powder showed 1.5 times the control value. The results reported the enhanced cell viability in 2D conditions, and 3D culture with bone tissue spheroids (viability was almost 150%). Furthermore, alkaline phosphatase activity (ALP) was tested in both monolayer and spheroid conditions, and the results indicated a boosted ALP activity in 2D and 3D condition (4 and 2 times the control value respectively). Additionally, nanocomposite powder extracts promoted the expression of both the COL1 and VEGF genes in 3D cultures (5 and 1.5 times the first day value respectively), showing the extract capability in accelerating bone spheroid formation. The current proof-of-concept study showed that 3D culture technology could provide a valuable way to understand the potential of tissue engineering before undertaking costly and harsh animal studies.

Simple Summary: Cholangiocarcinoma is a rare, aggressive, and heterogeneous malignancy of the bile duct. A typical feature of cholangiocarcinoma is that the cancer cells are embedded in a dense stroma. However, the functional role of the reactive tumor stroma has not been fully elucidated, which is certainly due to the lack of suitable in vitro models. Tumor stromal cells or tumor-associated fibroblasts play a central role in angiogenesis, metastasis, and the development of resistance to therapy in cholangiocarcinoma. We successfully isolated tumor cells and tumor-associated fibroblasts from a patient, generated cell lines, characterized the cells in detail, and established an innovative autologous tumor model. By detailed characterization of the tumor/tumor-associated fibroblast model we demonstrated that tumor cells interact with tumor-associated fibroblasts. This model can be used to study tumor stromal crosstalk, tumor angiogenesis and invasion, and the development of drug resistance. Cholangiocarcinoma (CCA) are characterized by their desmoplastic and hypervascularized tumor microenvironment (TME), which is mainly composed of tumor cells and cancer-associated fibroblasts (CAFs). CAFs play a pivotal role in general and CCA tumor progression, angiogenesis, metastasis, and the development of treatment resistance. To our knowledge, no continuous human in vivo-like co-culture model is available for research. Therefore, we aimed to establish a new model system (called MUG CCArly) that mimics the desmoplastic microenvironment typically seen in CCA. Proteomic data comparing the new CCA tumor cell line with our co-culture tumor model (CCTM) indicated a higher gene expression correlation of the CCTM with physiological CCA characteristics. A pro-angiogenic TME that is typically observed in CCA could also be better simulated in the CCTM group. Further analysis of secreted proteins revealed CAFs to be the main source of these angiogenic factors. Our CCTM MUG CCArly represents a new, reproducible, and easy-to-handle 3D CCA model for preclinical studies focusing on CCA-stromal crosstalk, tumor angiogenesis, and invasion, as well as the immunosuppressive microenvironment and the involvement of CAFs in the way that drug resistance develops. [ABSTRACT FROM AUTHOR]

• Primary human epithelial tissues are more reliable than cell lines to evaluate cystic fibrosis therapies. • Nasal, bronchial, and rectal epithelia have proven most useful in analyzing CFTR function and responses to cystic fibrosis therapeutics. • 2D and 3D patient-derived cell models are considered the most reliable disease models since they mimic the in vivo ion channel pathophysiology. • Personalized medicine approaches utilize advanced models based on patient-derived tissues to tailor therapies for specific patients to cure cystic fibrosis. Cystic fibrosis is caused by mutations in the cystic fibrosis transmembrane conductance regulator (CFTR) channel that perturb anion transport across the epithelia of the airways and other organs. To treat cystic fibrosis, strategies that target mutant CFTR have been developed such as correctors that rescue folding and enhance transfer of CFTR to the apical membrane, and potentiators that increase CFTR channel activity. While there has been tremendous progress in development and approval of CFTR therapeutics for the most common (F508del) and several other CFTR mutations, around 10-20% of people with cystic fibrosis have rare mutations that are still without an effective treatment. In the current decade, there was an impressive evolution of patient-derived cell models for precision medicine. In cystic fibrosis, these models have played a crucial role in characterizing the molecular defects in CFTR mutants and identifying compounds that target these defects. Cells from nasal, bronchial, and rectal epithelia are most suitable to evaluate treatments that target CFTR. In vitro assays using cultures grown at an air-liquid interface or as organoids and spheroids allow the diagnosis of the CFTR defect and assessment of potential treatment strategies. An overview of currently established cell culture models and assays for personalized medicine approaches in cystic fibrosis will be provided in this review. These models allow theratyping of rare CFTR mutations with available modulator compounds to predict clinical efficacy. Besides evaluation of individual personalized responses to CFTR therapeutics, patient-derived culture models are valuable for testing responses to developmental treatments such as novel RNA- and DNA-based therapies. [ABSTRACT FROM AUTHOR]

Cell therapies have the potential to improve reconstructive procedures for congenital craniofacial cartilage anomalies such as microtia. Adipose-derived stem cells (ADSCs) and auricular cartilage stem/progenitor cells (CSPCs) are promising candidates for cartilage reconstruction, but their successful use in the clinic will require the development of xeno-free expansion and differentiation protocols that can maximize their capacity for chondrogenesis. We assessed the behavior of human ADSCs and CSPCs grown either in qualified fetal bovine serum (FBS) or human platelet lysate (hPL), a xeno-free alternative, in conventional monolayer and 3-dimensional spheroid cultures. We show that CSPCs and ADSCs display greater proliferation rate in hPL than FBS and express typical mesenchymal stromal cell surface antigens in both media. When expanded in hPL, both cell types, particularly CSPCs, maintain a spindle-like morphology and lower surface area over more passages than in FBS. Both media supplements support chondrogenic differentiation of CSPCs and ADSCs grown either as monolayers or spheroids. However, chondrogenesis appears less ordered in hPL than FBS, with reduced co-localization of aggrecan and collagen type II in spheroids. hPL may be beneficial for the expansion of cells with chondrogenic potential and maintaining stemness, but not for their chondrogenic differentiation for tissue engineering or disease modeling. [ABSTRACT FROM AUTHOR]

Kidney disease is one of the leading causes of morbidity worldwide, emphasizing the importance for physiologically accurate disease models. With most of the approved renal drugs failing to perform as well in human clinical trials as they did in animal testing, it is imperative that new and improved human-based models are developed to test these potential therapeutics. One option is to use patient derived cell lines, grown in both two-dimensional (2D) and three-dimensional (3D) structures, known as spheroids and organoids. Despite their contributions to the field, the lack of physiological accuracy, including the absence of fluid flow, and mechanistic effects in these 2D and 3D models means there is still room for improvement. Organ-on-a-chip (OOAC) technology offers itself as a potential candidate model to overcome these limitations. Over recent years OOAC technology has grown in popularity, with multiple organ systems, including lung, liver, and kidney described in the literature. In this review, traditional human cellular based models, including monolayer, spheroid and organoid models will be discussed. Human kidney-on-a-chip models will also be discussed, while exploring the advantages and potential limitations of this rapidly emerging field for the study of human kidney disease and drug testing. [ABSTRACT FROM AUTHOR]

Aim: We investigated the delivery of sorafenib (SFB) to breast cancer spheroids by natural killer cell-derived exosomes (NK-Exos). Methods: SFB-NK-Exos were constructed by electroporation. Their antitumor effects were evaluated by methyl thiazolyl tetrazolium, acridine orange/ethidium bromide, 4′,6-diamidino-2-phenylindole, annexin/propidium iodide, scratch and migration assay, colony formation, RT-PCR, western blot and lipophagy tests. Result: The loading efficacy was 46.66%. SFB-NK-Exos-treated spheroids showed higher cytotoxic effects (33%) and apoptotic population (44.9%). Despite the reduction of SFB concentration in the SFB-NK-Exos formulation, similar cytotoxic effects to those of free SFB were observed. Increased intracellular trafficking, sustained release of the drug and selective inhibitory effects demonstrated efficient navigation. Conclusion: This is the first report for SFB loading into NK-Exos, which led to significant cytotoxic intensification against cancer cells. This study describes the delivery of an anticancer drug called sorafenib (SFB) to laboratory-grown spherical masses of cancer cells called spheroids. Saucer-like cellular structures called exosomes were used as drug-delivery tools. These exosomes were produced by a subgroup of immune cells called natural killer (NK) cells. NK cells are responsible for killing cancer cells. So, these exosomes share similar anticancer properties with NK cells. We wanted to test whether exosomes loaded with SFB would have better anticancer effects. Using different methods, SFB was loaded within the exosomes and delivered to the spheroids. The obtained results showed that a combination of exosomes and SFB could improve the targeting efficacy, reducing the side effects to the normal cells and allowing continuous release of the drug. The spheroids were killed with higher efficacy following this treatment. The combination of NK cell-derived exosomes and SFB could lead to better cytotoxicity against cancer cells. Therefore, this strategy could have better anticancer effects compared with SFB treatment alone. [ABSTRACT FROM AUTHOR]

This study aimed to develop an in vitro three-dimensional (3D) cell culture model of oral carcinogenesis for the rapid, scalable testing of chemotherapeutic agents. Spheroids of normal (HOK) and dysplastic (DOK) human oral keratinocytes were cultured and treated with 4-nitroquinoline-1-oxide (4NQO). A 3D invasion assay using Matrigel was performed to validate the model. RNA was extracted and subjected to transcriptomic analysis to validate the model and assess carcinogen-induced changes. The VEGF inhibitors pazopanib and lenvatinib were tested in the model and were validated by a 3D invasion assay, which demonstrated that changes induced by the carcinogen in spheroids were consistent with a malignant phenotype. Further validation was obtained by bioinformatic analyses, which showed the enrichment of pathways associated with hallmarks of cancer and VEGF signalling. Overexpression of common genes associated with tobacco-induced oral squamous cell carcinoma (OSCC), such as MMP1, MMP3, MMP9, YAP1, CYP1A1, and CYP1B1, was also observed. Pazopanib and lenvatinib inhibited the invasion of transformed spheroids. In summary, we successfully established a 3D spheroid model of oral carcinogenesis for biomarker discovery and drug testing. This model is a validated preclinical model for OSCC development and would be suitable for testing a range of chemotherapeutic agents. [ABSTRACT FROM AUTHOR]

Here we aimed to establish a simple detection method for detecting circulating tumor cells (CTCs) in the blood sample of colorectal cancer (CRC) patients using poly(2-methoxyethyl acrylate) (PMEA)-coated plates. Adhesion test and spike test using CRC cell lines assured efficacy of PMEA coating. A total of 41 patients with pathological stage II–IV CRC were enrolled between January 2018 and September 2022. Blood samples were concentrated by centrifugation by the OncoQuick tube, and then incubated overnight on PMEA-coated chamber slides. The next day, cell culture and immunocytochemistry with anti-EpCAM antibody were performed. Adhesion tests revealed good attachment of CRCs to PMEA-coated plates. Spike tests indicated that ~75% of CRCs from a 10-mL blood sample were recovered on the slides. By cytological examination, CTCs were identified in 18/41 CRC cases (43.9%). In cell cultures, spheroid-like structures or tumor-cell clusters were found in 18/33 tested cases (54.5%). Overall, CTCs and/or growing circulating tumor cells were found in 23/41 CRC cases (56.0%). History of chemotherapy or radiation was significantly negatively correlated with CTC detection (p = 0.02). In summary, we successfully captured CTCs from CRC patients using the unique biomaterial PMEA. Cultured tumor cells will provide important and timely information regarding the molecular basis of CTCs. [ABSTRACT FROM AUTHOR]

Introduction: Solid tumors consist of tumor cells associated with stromal and immune cells, secreted factors and extracellular matrix (ECM), which together constitute the tumor microenvironment. Among stromal cells, activated fibroblasts, known as cancer-associated fibroblasts (CAFs) are of particular interest. CAFs secrete a plethora of ECM components including collagen and modulate the architecture of the ECM, thereby influencing cancer cell migration. The characterization of the collagen fibre network and its space and time-dependent microstructural modifications is key to investigating the interactions between cells and the ECM. Developing image analysis tools for that purpose is still a challenge because the structural complexity of the collagen network calls for specific statistical descriptors. Moreover, the low signal-to-noise ratio of imaging techniques available for time-resolved studies rules out standard methods based on image segmentation. Methods: In this work, we develop a novel approach based on the stochastic modelling of the gel structure and on grey-tone image analysis. The method is then used to study the remodelling of a collagen matrix by migrating breast cancer-derived CAFs in a three-dimensional spheroid model of cellular invasion imaged by time-lapse confocal microscopy. Results: The structure of the collagen at the scale of a few microns consists in regions with high fibre density separated by depleted regions, which can be thought of as aggregates and pores. The approach developped captures this two-scale structure with a clipped Gaussian field model to describe the aggregates-and-pores large-scale structure, and a homogeneous Boolean model to describe the small-scale fibre network within the aggregates. The model parameters are identified by fitting the grey-tone histograms and correlation functions of the images. The method applies to unprocessed greytone images, and it can therefore be used with low magnification, noisy timelapse reflectance images. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells. Conclusion: We developed a novel and multidisciplinary image analysis approach to investigate the remodelling of fibrillar collagen in a 3D spheroid model of cellular invasion. The specificity of the method is that it applies to the unprocessed grey-tone images, and it can therefore be used with noisy timelapse reflectance images of non-fluorescent collagen. When applied to the CAF spheroid time-resolved images, the method reveals different matrix densification mechanisms for the matrix in direct contact or far from the cells. [ABSTRACT FROM AUTHOR]

Tissue organoids hold enormous potential as tools for a variety of applications, including disease modeling and drug screening. To effectively mimic the native tissue environment, it is critical to integrate a microvasculature with the parenchyma and stroma. In addition to providing a means to physiologically perfuse the organoids, the microvasculature also contributes to the cellular dynamics of the tissue model via the cells of the perivascular niche, thereby further modulating tissue function. In this review, we discuss current and developing strategies for vascularizing organoids, consider tissue-specific vascularization approaches, discuss the importance of perfusion, and provide perspectives on the state of the field. [ABSTRACT FROM AUTHOR]

Simple Summary: Organ-on-a-chip models, or organ chips, are tiny devices designed to accurately recreate the natural physiology and mechanical forces that cells experience in the human body. Similar to computer microchips, though carrying fluid through channels instead of an electric current, organ-chips are lined with living human cells and their tiny channels can reproduce blood and/or airflow just as in the human body. Their flexibility allows the chips to recreate breathing motions or undergo muscle contractions. This new and rapidly expanding field of research provides a unique opportunity to build models of human organs and to study how cancer cells develop and spread within them. As these chips are simpler and cheaper than animal models of cancer, they could significantly increase the speed of drug discovery and testing in cancer research. This exciting potential has led to the rapid development of this technology in the United Kingdom, with active research on a range of cancer types. This review covers the broad sweep of organ-chip research in the UK, and the network of researchers and companies being developed. Finally, it concludes with a perspective on the future directions in the field as researchers aim to bring about a leap forward in cancer therapies. Organ-on-chip systems are capable of replicating complex tissue structures and physiological phenomena. The fine control of biochemical and biomechanical cues within these microphysiological systems provides opportunities for cancer researchers to build complex models of the tumour microenvironment. Interest in applying organ chips to investigate mechanisms such as metastatsis and to test therapeutics has grown rapidly, and this review draws together the published research using these microfluidic platforms to study cancer. We focus on both in-house systems and commercial platforms being used in the UK for fundamental discovery science and therapeutics testing. We cover the wide variety of cancers being investigated, ranging from common carcinomas to rare sarcomas, as well as secondary cancers. We also cover the broad sweep of different matrix microenvironments, physiological mechanical stimuli and immunological effects being replicated in these models. We examine microfluidic models specifically, rather than organoids or complex tissue or cell co-cultures, which have been reviewed elsewhere. However, there is increasing interest in incorporating organoids, spheroids and other tissue cultures into microfluidic organ chips and this overlap is included. Our review includes a commentary on cancer organ-chip models being developed and used in the UK, including work conducted by members of the UK Organ-on-a-Chip Technologies Network. We conclude with a reflection on the likely future of this rapidly expanding field of oncological research. [ABSTRACT FROM AUTHOR]

Three-dimensional cell culture systems offer greater understanding of the complex human body structure than monolayer cell cultures. Spheroids, which are the most useful and controllable types of three-dimensional cell formations, are discussed in this review. Conventional spheroid fabrication methods have limitations for the mass production of uniformly sized spheroids, which hinders their further application. As an alternative, microfabrication methods have been proposed to overcome the drawbacks of existing methods. Microfabrication technologies include micropatterning, 3D bioprinting, and microfluidics. Microwell arrays and surface-modified micropatterns can be fabricated through micropatterning methods, and these scaffolds result in the mass production of spheroids with size uniformity. 3D bioprinting technology enables uniformly sized spheroid production at desired locations, and microfluidics allows production of uniform size-controlled spheroids in a large quantity Recently, efforts have been made to apply 3D spheroid culture systems to regenerative medicine, the study of the tumor microenvironment, drug screening, and organoid fabrication. The 3D spheroid system is an attractive substitute for overcoming the limitations of the conventional 2D culture platform, which cannot precisely imitate in vivo physiological environments. Microfabrication methods for spheroids enhance the effectiveness of spheroid formation, allowing for mass production, size control, and spheroid localization. Microfabrication methods have remarkable potential for spheroid utilization in the biomedical field. [ABSTRACT FROM AUTHOR]

Membrane type 1 matrix metalloproteinase (MT1-MMP) has been shown to be crucial for tumor angiogenesis, invasion, and metastasis, and thus MT1-MMP is a high priority target for potential cancer therapies. To properly evaluate MT1-MMP inhibitors, a screening protocol is desired by which enzyme activity can be quantified in a tumor microenvironment-like model system. In the present study, we applied a fluorogenic, collagen model triple-helical substrate to quantify MT1-MMP activity for tumor spheroids embedded in a collagen hydrogel. The substrate was designed to be MT1-MMP selective and to possess fluorescent properties compatible with cell-based assays. The proteolysis of the substrate correlated to glioma spheroid invasion. In turn, the application of either small molecule or protein-based MMP inhibitors reduced proteolytic activity and glioma spheroid invasion. The presence of MT1-MMP in glioma spheroids was confirmed by western blotting. Thus, spheroid invasion was dependent on MT1-MMP activity, and inhibitors of MT1-MMP and invasion could be conveniently screened in a high-throughput format. The combination of the fluorogenic, triple-helical substrate, the three-dimensional tumor spheroids embedded in collagen, and Hit-Pick software resulted in an easily adaptable in vivo-like tumor microenvironment for rapidly processing inhibitor potential for anti-cancer use. [ABSTRACT FROM AUTHOR]

Background: Ovarian cancer cells aggregate during or after exfoliation from the primary tumor to form threedimensional spheroids. Spheroid formation provides a survival advantage during peritoneal dissemination in nutrient and oxygen-depleted conditions which is accompanied by a suppressed metabolic phenotype and fragmented mitochondria. Upon arrival to their metastatic sites, spheroids adhere to peritoneal organs and transition to a more epithelial phenotype to support outgrowth and invasion. In this study, we investigated the plasticity of mitochondrial morphology, dynamics, and function upon adhesion. Methods: Using our slow-developing (MOSE-L) and fast-developing (MOSE-LTICv) ovarian cancer models, we mimicked adhesion and reoxygenation conditions by plating the spheroids onto tissue culture dishes and changing culture conditions from hypoxia and low glucose to normoxia with high glucose levels after adhesion. We used Western Blot, microscopy and Seahorse analyses to determine the plasticity of mitochondrial morphology and functions upon adhesion, and the impact on proliferation and invasion capacities. Results: Independent of culture conditions, all spheroids adhered to and began to grow onto the culture plates. While the bulk of the spheroid was unresponsive, the mitochondrial morphology in the outgrowing cells was indistinguishable from cells growing in monolayers, indicating that mitochondrial fragmentation in spheroids was indeed reversible. This was accompanied by an increase in regulators of mitobiogenesis, PGC1a, mitochondrial mass, and respiration. Reoxygenation increased migration and invasion in both cell types but only the MOSE-L responded with increased proliferation to reoxygenation. The highly aggressive phenotype of the MOSE-LTICv was characterized by a relative independence of oxygen and the preservation of higher levels of proliferation, migration and invasion even in limiting culture conditions but a higher reliance on mitophagy. Further, the outgrowth in these aggressive cells relies mostly on proliferation while the MOSE-L cells both utilize proliferation and migration to achieve outgrowth. Suppression of proliferation with cycloheximide impeded aggregation, reduced outgrowth and invasion via repression of MMP2 expression and the flattening of the spheroids. Discussion: Our studies indicate that the fragmentation of the mitochondria is reversible upon adhesion. The identification of regulatory signaling molecules and pathways of these key phenotypic alterations that occur during primary adhesion and invasion is critical for the identification of druggable targets for therapeutic intervention to prevent aggressive metastatic disease. [ABSTRACT FROM AUTHOR]

Spheroids are expected to aid the establishment of an in vitro-based cell culture system that can realistically reproduce cellular dynamics in vivo. We developed a fluoropolymer scaffold with an extracellular matrix (ECM) dot array and confirmed the possibility of mass-producing spheroids with uniform dimensions. Controlling the quality of ECM dots is important as it ensures spheroid uniformity, but issues such as pattern deviation and ECM drying persist in the conventional microstamping method. In this study, these problems were overcome via ECM dot printing using a resin mask with dot-patterned holes. For dot diameters of φ 300 μm, 400 μm, and 600 μm, the average spheroid diameters of human iPS cells (hiPSCs) were φ 260.8 μm, 292.4 μm, and 330.7 μm, respectively. The standard deviation when each average was normalized to 100 was 14.1%. A high throughput of 89.9% for colony formation rate to the number of dots and 89.3% for spheroid collection rate was achieved. The cells proliferated on ECM dots, and the colonies could be naturally detached from the scaffold without the use of enzymes, so there was almost no stimulation of the cells. Thus, the undifferentiated nature of hiPSCs was maintained until day 4. Therefore, this method is expected to be useful in drug discovery and regenerative medicine. [ABSTRACT FROM AUTHOR]

Introduction: Cholangiocarcinoma (CCA) is difficult to cure due to its ineffective treatment and advanced stage diagnosis. Thoroughly mechanistic understandings of CCA pathogenesis crucially help improving the treatment success rates. Using three-dimensional (3D) cell culture platform offers several advantages over a traditional two-dimensional (2D) culture as it resembles more closely to in vivo tumor. Methods: Here, we aimed to establish the 3D CCA spheroids with lowly (KKU-100) and highly (KKU-213A) metastatic potentials to investigate the CCA migratory process and its EMT-associated galectin-3 in the 3D setting. Results and discussion: Firstly, the growth of lowly metastatic KKU-100 cells was slower than highly metastatic KKU-213A cells in both 2D and 3D systems. Hollow formation was observed exclusively inside the KKU-213A spheroids, not in KKU-100. Additionally, the migration activity of KKU-213A cells was higher than that of KKU-100 cells in both 2D and 3D systems. Besides, altered expression of galectin-3 were observed across all CCA culture conditions with substantial relocalization from inside the 2D cells to the border of spheroids in the 3D system. Notably, the CCA migration was inversely proportional to the galectin-3 expression in the 3D culture, but not in the 2D setting. This suggests the contribution of culture platforms to the alternation of the CCA cell migration process. Conclusions: Thus, our data revealed that 3D culture of CCA cells was phenotypically distinct from 2D culture and pointed to the superiority of using the 3D culture model for examining the CCA cellular mechanisms, providing knowledges that are better correlated with CCA phenotypes in vivo. [ABSTRACT FROM AUTHOR]

Background: Head and neck squamous cell carcinoma (HNSCC) is one of the leading causes of cancer-related death worldwide. Surgical resection, radiation and chemotherapy are the mainstay of HNSCC treatment but are often unsatisfactory. Cisplatin is a commonly used chemotherapy in HNSCC; however, cisplatin resistance is a major cause of relapse and death. The 78-kD glucose-regulated protein (GRP78) is the master regulator of the unfolded protein response (UPR) and is implicated in therapeutic resistance in cancer. The role of GRP78 in cisplatin resistance in HNSCC remains unclear. YUM70 is a newly discovered hydroxyquinoline analogue and found to be an inhibitor of GRP78. The effect of YUM70 in HNSCC cell lines is unknown. Method: Knockdown of GRP78 by siRNAs was performed to investigate the effect of GRP78 reduction in endoplasmic reticulum (ER)-stress induced and general apoptosis. Western blots examining apoptotic markers were performed on three HPV-negative HNSCC cell lines. WST-1 assay was performed to determine cell viability. In reverse, we utilized AA147, an ER proteostasis regulator to upregulate GRP78, and apoptotic markers and cell viability were determined. To test the ability of YUM70 to reverse cisplatin resistance, cisplatin-resistant HNSCC cell lines were generated by prolonged, repeated exposure to increasing concentrations of cisplatin. Colony formation assay using the cisplatin-resistant HNSCC cell line was performed to assess the in vitro reproductive cell survival. Furthermore, to test the ability of YUM70 to reverse cisplatin resistance in a physiologically relevant system, we subjected the 3D spheroids of the cisplatin-resistant HNSCC cell line to cisplatin treatment with or without YUM70 and monitored the onset of apoptosis. Results: Reduction of GRP78 level induced HNSCC cell death while GRP78 upregulation conferred higher resistance to cisplatin. Combined cisplatin and YUM70 treatment increased apoptotic markers in the cisplatin-resistant HNSCC cell line, associating with reduced cell viability and clonogenicity. The combination treatment also increased apoptotic markers in the 3D spheroid model. Conclusion: The GRP78 inhibitor YUM70 reduced HNSCC cell viability and resensitized cisplatin-resistant HNSCC cell line in both 2D and 3D spheroid models, suggesting the potential use of YUM70 in the treatment of HNSCC, including cisplatin-resistant HNSCC. [ABSTRACT FROM AUTHOR]

The liver produces and stores various nutrients that are necessary for the body and serves as a chemical plant, metabolizing carbohydrates, fats, hormones, vitamins, and minerals. It is also a vital organ for detoxifying drugs and exogenous harmful substances. Culturing liver cells in vitro under three-dimensional (3D) conditions is considered a primary mechanism for liver tissue engineering. The 3D cell culture system is designed to allow cells to interact in an artificially created environment and has the advantage of mimicking the physiological characteristics of cells in vivo. This system facilitates contact between the cells and the extracellular matrix. Several technically different approaches have been proposed, including bioreactors, chips, and plate-based systems in fluid or static media composed of chemically diverse materials. Compared to conventional two-dimensional monolayer culture in vitro models, the ability to predict the function of the tissues, including the drug metabolism and chemical toxicity, has been enhanced by developing three-dimensional liver culture models. This review discussed the methodology of 3D cell cultures and summarized the advantages of an in vitro liver platform using 3D culture technology. [ABSTRACT FROM AUTHOR]

Objective. To observe changes in the shapes and growth patterns of melanocytes (MCs) after receiving two long-term trypsinization (LTT) treatments for 2 hours each. Methods. Epidermal primary cells were obtained from foreskin tissues, which derived from routine circumcisions. Antibodies specific for keratin 15 (K15) and melanosomes (HMB-45) were used to identify keratinocytes (KCs) and MCs, respectively. MCs were purified via differential trypsinization, and then continued to be cultivated. When they became 70% confluent, the MCs were treated with LTT and were subcultured. After about 1 week, the above treatment was repeated. Changes in the morphologies and growth patterns of MCs were observed daily, were photographed and analyzed. Results. The results of immunofluorescence staining showed MCs presenting with dendrites and KCs with cobblestone-shapes coexisting in the epidermal cultures. MCs were purified by differential trypsinization and appeared as dendritic, in monolayer growth, with a doubling time of 3-5 d. After 2 h of LTT, those MCs proliferated more quickly with a doubling time of 2-3 d. Meanwhile, the number and length of dendrites were reduced, most of MCs were bipolar, and a few had three dendrites. After the second LTT, the MCs became short rod-shaped or fusiform, with a doubling time of less than 2 d. Some aggregates or spheroids of MCs gradually appeared and increased in size over the time of culture. Each MC spheroid (MS) contained 3-30 MCs, with various morphologies and sizes within the same spheroid. When MSs were picked up and re-seeded, the dendritic cells migrated out and expanded surrounding the spheroids. They proliferated rapidly, grew in a monolayer, and were morphologically similar to primary MCs. Conclusion. LTT reduced the number of dendrites and shortened the doubling time of MCs. Two repeat treatments of LTT can induce the formation of MSs. [ABSTRACT FROM AUTHOR]

BACKGROUND: Mesenchymal stem cell (MSC)-based therapies offer potential for bone repair. MSC spheroid cultures may harbor enhanced therapeutic potential over MSC monolayers through increased secretion of trophic factors. However, the impact of spheroid size on trophic factor expression is unclear. OBJECTIVE: We investigated the effect of spheroid size on trophic factor-related gene expression. METHODS: KUM10, a murine MSC line was used. RNA-seq was used to screen the transcriptional profiles of MSC monolayer and spheroid cultures. Differentially expressed genes identified in RNA-seq were evaluated by q-PCR in cultures of 5 × 104 (S group), 5 × 105 (M group), 5 × 106 (L group) cells/well. RESULTS: Comparison of expression levels between KUM10 monolayer and spheroid cultures identified 2140 differentially expressed genes, of which 1047 were upregulated and 1093 were downregulated in KUM10 spheroids. Among these, 12 upregulated genes (Bmp2, Fgf9, Fgf18, Ngf, Pdgfa, Pdgfb, Tgfb1, Vegfa, Vegfc, Wnt4, Wnt5a, Wnt10a) were associated with secretory growth factors. Of these, expression of Fgf9, Fgf18, Vegfa and Vegfc was elevated in the L group, and Pdgfb and Tgfb1 was elevated in the S group. CONCLUSIONS: Spheroid size may impact trophic factor expression. Our results will be useful for future studies assessing the utility of MSC spheroids for treating bone injury. [ABSTRACT FROM AUTHOR]

Introduction: One of the key factors that may influence the therapeutic potential of mesenchymal stem/stromal cells (MSCs) is their metabolism. The switch between mitochondrial respiration and glycolysis can be affected by many factors, including the oxygen concentration and the spatial form of culture. This study compared the metabolic features of adipose-derived mesenchymal stem/stromal cells (ASCs) and dedifferentiated fat cells (DFATs) cultivated as monolayer or spheroid culture under 5% O2 concentration (physiological normoxia) and their impact on MSCs therapeutic abilities. Results: We observed that the cells cultured as spheroids had a slightly lower viability and a reduced proliferation rate but a higher expression of the stemness-related transcriptional factors compared to the cells cultured in monolayer. The three-dimensional culture form increased mtDNA content, oxygen consumption rate (OCR) and extracellular acidification rate (ECAR), especially in DFATs-3D population. The DFATs spheroids also demonstrated increased levels of Complex V proteins and higher rates of ATP production. Moreover, increased reactive oxygen species and lower intracellular lactic acid levels were also found in 3D culture. Conclusion: Our results may suggest that metabolic reconfiguration accompanies the transition from 2D to 3D culture and the processes of both mitochondrial respiration and glycolysis become more active. Intensified metabolism might be associated with the increased demand for energy, which is needed to maintain the expression of pluripotency genes and stemness state. [ABSTRACT FROM AUTHOR]

Small cell lung cancer (SCLC) is frequently disseminated and has a dismal prognosis with survival times of approximately two years. This cancer responds well to initial chemotherapy but recurs within a short time as a globally chemoresistant tumor. Circulating tumor cells (CTCs) are held responsible for metastasis, the extremely high numbers of these cells in advanced SCLC allowed us to establish several permanent CTC cell lines. These CTCs are distinguished by the spontaneous formation of large spheroids, termed tumorospheres, in regular tissue culture. These contain quiescent and hypoxic cells in their interior and are associated with high chemoresistance compared to single cell cultures. Nine CTC lines were compared for their expression of 84 proteins associated with cancer either as single cells or in the form of tumorospheres in Western blot arrays. With the exception of the UHGc5 line, all other CTC lines express EpCAM and lack a complete EpCAM-negative, vimentin-positive epithelial-mesenchymal transition (EMT) phenotype. Upon formation of tumorospheres the expression of EpCAM, that mediates cell-cell adhesion is markedly upregulated. Proteins such as E-Cadherin, p27 KIP1, Progranulin, BXclx, Galectin-3, and Survivin showed variable changes for the distinct CTC cell lines. In conclusion, EpCAM presents the most critical marker for individual SCLC CTCs and the assembly of highly chemoresistant tumorospheres. [ABSTRACT FROM AUTHOR]

In recent years, three-dimensional (3D) cell culture has been attracting attention as a cell culture model that mimics an environment closer to that of a living organism. It is known that there is a close relationship between cell nuclear shape and cellular function, which highlights the importance of cell nucleus shape analysis in the 3D culture. On the other hand, it is difficult to observe the cell nuclei inside the 3D culture models because the penetration depth of the laser light under a microscope is limited. In this study, we adopted an aqueous iodixanol solution to the 3D osteocytic spheroids derived from mouse osteoblast precursor cells to make the spheroids transparent for 3D quantitative analysis. With a custom-made image analysis pipeline in Python, we found that the aspect ratio of the cell nuclei near the surface of the spheroid was significantly greater than that at the center, suggesting that the nuclei on the surface were deformed more than those at the center. The results also quantitatively showed that the orientation of nuclei in the center of the spheroid was randomly distributed, whereas those on the surface of the spheroid were oriented parallel to the surface of the spheroid. Our 3D quantitative method with an optical clearing technique will contribute to the 3D culture models including various organoid models to elucidate the nuclear deformation during the development of the organs. Insight box Although 3D cell culture has been a powerful tool in the fields of fundamental biology and tissue engineering, it raises the demand for quantification techniques for cell nuclear morphology in the 3D culture model. In this study, we attempted to optically clear a 3D osteocytic spheroid model using iodixanol solution for the nuclear observation inside the spheroid. Moreover, using a custom-made image analysis pipeline in Python, we successfully quantified the nuclear morphology regarding aspect ratio and orientation. Our quantitative method with the optical clearing technique will contribute to the 3D culture models such as various organoid models to elucidate the nuclear deformation during the development of the organs. [ABSTRACT FROM AUTHOR]

Managing metastasis at the early stage and detecting and treating submillimeter tumors at early metastasis are crucial for improving cancer prognosis. Angiogenesis is a critical target for developing drugs to detect and inhibit submillimeter tumor growth; however, drug development remains challenging because there are no suitable models for observing the submillimeter tumor mass and the surrounding blood vessels in vivo. We have established a xenograft subcutaneous submillimeter tumor mouse model with HT-29-RFP by transplanting a single spheroid grown on radiation-crosslinked gelatin hydrogel microwells. Here, we developed an in vivo dual-observation method to observe the submillimeter tumor mass and tumor-surface blood vessels using this model. RFP was detected to observe the tumor mass, and a fluorescent angiography agent FITC-dextran was administered to observe blood vessels via stereoscopic fluorescence microscopy. The anti-angiogenesis agent regorafenib was used to confirm the usefulness of this method. This method effectively detected the submillimeter tumor mass and tumor-surface blood vessels in vivo. Regorafenib treatment revealed tumor growth inhibition and angiogenesis downregulation with reduced vascular extremities, segments, and meshes. Further, we confirmed that tumor-surface blood vessel areas monitored using in vivo dual-observation correlated with intratumoral blood vessel areas observed via fluorescence microscopy with frozen sections. In conclusion, this method would be useful in developing anti-angiogenesis agents against submillimeter tumors.

Microfluidic devices, which miniaturize cell culture and chemical experiments from lab-scale to microchip dimensions, have gained significant attention in recent years. Extensive research has been conducted on microfluidic mixers, which facilitate the mixing and agitation of chemicals. The “Sidewall-Driven Micromixer” that we are currently developing employs a unique mechanism; it induces a swirling flow within the main chamber by vibrating the silicone wall situated between the main and driving chambers using pressure fluctuations. In an earlier study, we found that Sidewall-Driven Micromixers of a size suitable for small cells could indeed produce this swirling flow. Furthermore, we successfully established concentration gradients within each mixer. However, when attempting to upscale the mixer while maintaining conventional proportions to accommodate larger cell aggregates such as spheroids, the desired swirling flow was not achieved. To address this challenge, we made adjustments to the wall dimensions, aiming to amplify wall deformation and thereby enhance the mixer’s driving force. Concurrently, we modified the mixer’s shape to ensure that the increased wall deformation would not hinder the fluid flow. These alterations not only improved the mixer’s performance but also provided valuable insights for positioning the mixer’s neck channel, considering the extent of wall deformation.

Overcoming drug resistance and specifically targeting cancer stem cells (CSCs) are critical challenges in improving cancer therapy. Nowadays, the use of novel and native medicinal plants can provide new sources for further investigations for this purpose. The aim of this study was to assess the potential of S. bachtiarica, an endemic plant with diverse medicinal applications, in suppressing and targeting cancer and cancer stem cells in glioblastoma and breast cancer. The effect of S. bachtiarica on viability, migration, invasion, and clonogenic potential of MDAMB-231 and U87-MG cells was assessed in both two- and three-dimensional cell culture models. Additionally, we evaluated its effects on the self-renewal capacity of mammospheres. The experimental outcomes indicated that S. bachtiarica decreased the viability and growth rate of cells and spheroids by inducing apoptosis and inhibited colony formation, migration, and invasion of cells and spheroids. Additionally, colony and sphere-forming ability, as well as the expression of genes associated with EMT and stemness were reduced in mammospheres treated with S. bachtiarica. In conclusion, this study provided valuable insights into the anti-cancer effects of S. bachtiarica, particularly in relation to breast CSCs. Therefore, S. bachtiarica may be a potential adjuvant for the treatment of cancer.

Functionally enhanced mesenchymal stromal cells participate in the repair of intervertebral disc. This study aimed to assess the safety and tolerability of intradiscal administration of matrilin-3-primed adipose-derived stromal cell (ASC) spheroids with hyaluronic acid (HA) in patients with chronic discogenic low back pain (LBP). In this single-arm, open-label phase I clinical trial, eight patients with chronic discogenic LBP were observed over 6 months. Each patient underwent a one-time intradiscal injection of 1 mL of 6.0 × 106 cells/disc combined with HA under real-time fluoroscopic guidance. Safety and feasibility were gauged using Visual Analogue Scale (VAS) pain and Oswestry Disability Index (ODI) scores and magnetic resonance imaging. All participants remained in the trial, with no reported adverse events linked to the procedure or stem cells. A successful outcome-marked by a minimum 2-point improvement in the VAS pain score and a 10-point improvement in ODI score from the start were observed in six participants. Although the modified Pfirrmann grade remained consistent across all participants, radiological improvements were evident in four patients. Specifically, two patients exhibited reduced high-intensity zones while another two demonstrated decreased disc protrusion. In conclusion, the intradiscal application of matrilin-3-primed ASC spheroids with HA is a safe and feasible treatment option for chronic discogenic LBP.

Cancer stem cells (CSCs) are considered to be responsible for recurrence, metastasis, and resistance to treatment in many types of cancers; therefore, new treatment strategies targeting CSCs are attracting attention. In this study, we fabricated a polyethylene glycol-tagged microwell device that enabled spheroid formation from human oral squamous carcinoma cells. HSC-3 and Ca9-22 cells cultured in the microwell device aggregated and generated a single spheroid per well within 24–48 h. The circular shape and smooth surface of spheroids were maintained for up to five days, and most cells comprising the spheroids were Calcein AM-positive viable cells. Interestingly, the mRNA expression of CSC markers (Cd44, Oct4, Nanog, and Sox2) were significantly higher in the spheroids than in the monolayer cultures. CSC marker-positive cells were observed throughout the spheroids. Moreover, resistance to cisplatin was enhanced in spheroid-cultured cells compared to that in the monolayer-cultured cells. Furthermore, some CSC marker genes were upregulated in HSC-3 and Ca9-22 cells that were outgrown from spheroids. In xenograft model, the tumor growth in the spheroid implantation group was comparable to that in the monolayer culture group. These results suggest that our spheroid culture system may be a high-throughput tool for producing uniform CSCs in large numbers from oral cancer cells.

A basic process in cancer is the breaching of basement-membrane barriers to permit tissue invasion. Cancer cells can use proteases and physical mechanisms to produce initial holes in basement membranes, but how cells squeeze through this barrier into matrix environments is not well understood. We used a 3D invasion model consisting of cancer-cell spheroids encapsulated by a basement membrane and embedded in collagen to characterize the dynamic early steps in cancer-cell invasion across this barrier. We demonstrate that certain cancer cells extend exceptionally long (~30–100 μm) protrusions through basement membranes via actin and microtubule cytoskeletal function. These long protrusions use integrin adhesion and myosin II-based contractility to pull cells through the basement membrane for initial invasion. Concurrently, these long, organelle-rich protrusions pull surrounding collagen inward while propelling cancer cells outward through perforations in the basement-membrane barrier. These exceptionally long, contractile cellular protrusions can facilitate the breaching of the basement-membrane barrier as a first step in cancer metastasis.