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Tumor-associated macrophages (TAMs) constitute 50–80% of stromal cells in most solid tumors with high mortality and poor prognosis. Tumor-infiltrating dendritic cells (TIDCs) and TAMs are key components mediating immune responses within the tumor microenvironment (TME). Considering their refractory properties, simultaneous remodeling of TAMs and TIDCs is a potential strategy of boosting tumor immunity and restoring immunosurveillance. In this study, mannose-decorated poly(lactic- co -glycolic acid) nanoparticles loading with R848 (Man-pD-PLGA-NP@R848) were prepared to dually target TAMs and TIDCs for efficient tumor immunotherapy. The three-dimensional (3D) cell culture model can simulate tumor growth as influenced by the TME and its 3D structural arrangement. Consequently, cancer spheroids enriched with tumor-associated macrophages (TAMs) were fabricated to assess the therapeutic effectiveness of Man-pD-PLGA-NP@R848. In the TME, Man-pD-PLGA-NP@R848 targeted both TAMs and TIDCs in a mannose receptor-mediated manner. Subsequently, Man-pD-PLGA-NP@R848 released R848 to activate Toll-like receptors 7 and 8, following dual-reprograming of TIDCs and TAMs. Man-pD-PLGA-NP@R848 could uniquely reprogram TAMs into antitumoral phenotypes, decrease angiogenesis, reprogram the immunosuppressive TME from "cold tumor" into "hot tumor", with high CD4+ and CD8+ T cell infiltration, and consequently hinder tumor development in B16F10 tumor-bearing mice. Therefore, dual-reprograming of TIDCs and TAMs with the Man-pD-PLGA-NP@R848 is a promising cancer immunotherapy strategy. [Display omitted] • 3D spheroid (+TAM) models mimic the tumor models in clinical cancer patients. • The more M2-TAMs are in TME, the more macrophages are polarized to M1 phenotype. • Man-pD-PLGA-NP@R848 activates CD4+ and CD8+ T lymphocytes in TILs. • Man-pD-PLGA-NP@R848 stimulates and enhances the tumor-infiltrated cytokines level. • Man-pD-PLGA-NP@R848 as intratumoral immunotherapy to modulate tumor into hot state. [ABSTRACT FROM AUTHOR]

The development of bio-three-dimensional (bio-3D) printers has led to significant advances in regenerative medicine. Three-dimensional constructs, including spheroids, are maintained by extracellular matrix proteins secreted by cells so that the cells can be cultured in conditions closer to the physiological environment. This study aimed to create a useful 3D construct as a model of the dentin-pulp complex. We examined the expression patterns of extracellular matrix proteins and cell proliferation areas in a 3D construct created using O9-1 cells derived from cranial neural crest cells of mice. The 3D construct was created by sticking the spheroid cultures onto a needle array using a bio-3D printer. Cell proliferation areas along with characteristic expression of tenascin C and DMP1 were evaluated. The expression of tenascin C and DMP1 was significantly enhanced in the spheroids compared to that in two-dimensional cultures. Moreover, cell proliferation regions and tenascin C expression were confirmed in the outer layer of spheroids in the embryonic stem cell medium, with insignificant DMP1 expression being observed. Interestingly, in a 3D construct cultured in calcification-induction medium, DMP1 expression was promoted, and DMP1-positive cells existed in the outermost layer without overlapping with tenascin C expression. The extracellular matrix proteins, tenascin C and DMP1, were expressed in a polarized manner in spheroids and 3D constructs, similar to the findings in the dental papilla. Therefore, these 3D constructs show potential as artificial models for studying odontogenesis. [ABSTRACT FROM AUTHOR]

Most in vitro models of oviduct epithelial cells (OEC) used thus far to gain insights into embryo–maternal communication induce cell dedifferentiation or are technically challenging. Moreover, although the presence of developing embryos has been shown to alter gene expression in OEC, the effect of embryos on OEC physiology remains largely unknown. Here, we propose a model based on bovine oviduct epithelial spheroids (OES) with specific shape and diameter (100–200 μm) criteria. The aims of this study were to i) determine the appropriate culture conditions of bovine OES cultured in suspension by evaluating their morphology, total cell number, viability, and activity of ciliated cells; ii) monitor gene expression in OES at the time of their formation (day 0) and over the 10 days of culture; and iii) test whether the vicinity of developing embryos affects OES quality criteria. On day 10, the proportions of vesicle-shaped OES (V-OES) were higher in M199/500 (500 μl of HEPES-buffered TCM-199) and synthetic oviduct fluid (SOF)/25 (25-μL droplet of SOF medium under mineral oil) than in M199/25 (25-μL droplet of M199 under mineral oil). The proportion of viable cells in V-OES was not affected by culture conditions and remained high (>80%) through day 10. The total number of cells per V-OES decreased over time except in SOF/25, while the proportions of ciliated cells increased over time in M199/500 but decreased in M199/25 and SOF/25. The movement amplitude of OES in suspension decreased over time under all culture conditions. Moreover, the gene expression of ANXA1 , ESR1 , HSPA8 , and HSPA1A in OES remained stable during culture, while that of PGR and OVGP1 decreased from day 0 to day 10. Last, the co-culture of developing embryos with OES in SOF/25 increased the rates of blastocysts on days 7 and 8 compared to embryos cultured alone, and increased the proportion of V-OES compared to OES cultured alone. In conclusion, M199/500 and SOF/25 provided the optimal conditions for the long-time culture of OES. The supporting effect of OES on embryo development and of developing embryos on OES morphology was evidenced for the first time. Altogether, these results point OES as an easy-to-use, standardizable, and physiological model to study embryo–maternal interactions in cattle. • Oviduct epithelial spheroids (OES) were obtained from isthmic mucosa fragments. • Large volume of M199 and droplets of SOF offered good culture conditions for OES. • OES expressed oviduct epithelial cell markers during 10 days in culture. • OES supported embryo development in SOF medium. • Embryo supported OES morphology in SOF medium. [ABSTRACT FROM AUTHOR]

Stem cell therapy has emerged as a promising regenerative medicine strategy but is limited by poor cell survival, leading to low therapeutic outcomes. We developed cell spheroid therapeutics to overcome this limitation. We utilized solid-phase FGF2 to form functionally enhanced cell spheroid-adipose derived (FECS-Ad), a type of cell spheroid that preconditions cells with intrinsic hypoxia to increase the survival of transplanted cells. We demonstrated an increase in hypoxia-inducible factor 1-alpha (HIF-1α) levels in FECS-Ad, which led to the upregulation of tissue inhibitor of metalloproteinase 1 (TIMP1). TIMP1 enhanced the survival of FECS-Ad, presumably through the CD63/FAK/Akt/Bcl2 anti-apoptotic signaling pathway. Cell viability of transplanted FECS-Ad was reduced by TIMP1 knockdown in an in vitro collagen gel block and a mouse model of critical limb ischemia (CLI). TIMP1 knockdown in FECS-Ad inhibited angiogenesis and muscle regeneration induced by FECS-Ad transplanted into ischemic mouse tissue. Genetic overexpression of TIMP1 in FECS-Ad further promoted the survival and therapeutic efficacy of transplanted FECS-Ad. Collectively, we suggest that TIMP1 acts as a key survival factor to improve the survival of transplanted stem cell spheroids, which provides scientific evidence for enhanced therapeutic efficacy of stem cell spheroids, and FECS-Ad as a potential therapeutic agent to treat CLI. We used FGF2-tethered substrate platform to form adipose-derived stem cell spheroids, as we named as functionally enhanced cell spheroid-adipose derived (FECS-Ad). In this paper, we showed that intrinsic hypoxia of spheroids upregulated expression of HIF-1α, which in turn upregulated expression of TIMP1. Our paper highlights TIMP1 as a key survival factor to improve survival of transplanted stem cell spheroids. We believe that our study has a very strong scientific impact as extending transplantation efficiency is essential for successful stem cell therapy. [Display omitted] [ABSTRACT FROM AUTHOR]

Cellular spheroids are aggregates of cells that are being explored to address fundamental biological questions and as building blocks for engineered tissues. Spheroids possess distinct advantages over cellular monolayers or cell encapsulation in 3D natural and synthetic hydrogels, including direct cell-cell interactions and high cell densities, which better mimic aspects of many tissues. Despite these advantages, spheroid cultures often exhibit uncontrollable growth and may be too simplistic to mimic complex tissue structures. To address this, biomaterials are being leveraged to further expand the use of cellular spheroids for biomedical applications. In this review, we provide an overview of recent studies that utilize engineered biomaterials to guide spheroid formation and function , as well as their fabrication into tissues for use as tissue models and for therapeutic applications. First, we describe biomaterial strategies that allow the high-throughput fabrication of homogeneously-sized spheroids. Next, we summarize how engineered biomaterials are introduced into spheroid cultures either internally as microparticles or externally as hydrogel microenvironments to influence spheroid behavior (e.g., differentiation, fusion). Lastly, we discuss a variety of biofabrication strategies (e.g., 3D bioprinting, melt electrowriting) that have been used to develop macroscale tissue models and implantable constructs through the guided assembly of spheroids. Overall, the goal of this review is to provide a summary of how biomaterials are currently being engineered and leveraged to support spheroids in biomedical applications, as well as to provide a future outlook of the field. Cellular spheroids are becoming increasingly used as in vitro tissue models or as 'building blocks' for tissue engineering and repair strategies. Engineered biomaterials and their processing through biofabrication approaches are being leveraged to structurally support and guide spheroid processes. This review summarizes current approaches where such biomaterials are being used to guide spheroid formation, function , and fabrication into tissue constructs. As the field is rapidly expanding, we also provide an outlook on future directions and how new engineered biomaterials can be implemented to further the development of biofabricated spheroid-based tissue constructs. [Display omitted] [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]

Photodynamic therapy (PDT) is a medical radio chemotherapeutic method that uses light, photosensitizing agents, and oxygen to produce cytotoxic compounds, which eliminate malignant cells. Recently, Microfluidic systems have been used to analyse photosensitizers (PSs) due to their potential to replicate in vivo environments. While prior studies have established a strong correlation between reacted singlet oxygen concentration and PDT-induced cellular death, the effects that the ambient fluid flow might have on the concentration of oxygen and PS have been disregarded in many, which limits the reliability of the results. Herein, we coupled the transport of oxygen and PS throughout the ambient medium and within the spheroidal multicellular aggregate to initially study the profiles of oxygen and PS concentration alongside PDT-induced cellular death throughout the spheroid before and after radiation. The attained results indicate that the PDT-induced cellular death initiates on the surface of the spheroids and subsequently spreads to the neighbouring regions, which is in great accordance with experimental results. Afterward, the effects that drug-light interval (DLI), fluence rate, PS composition, microchannel height, and inlet flow rate have on the therapeutic outcomes are studied. The findings show that adequate DLI is critical to ensure uniform distribution of PS throughout the medium, and a value of 5 h was found to be sufficient. The composition of PS is critical, as ALA-PpIX induces earlier cell death but accelerates oxygen consumption, especially in the outer layers, depriving the inner layers of oxygen necessary for PDT, which in turn disrupts and prolongs the exposure time compared to mTHPC and Photofrin. Despite the fluence rate directly influencing the singlet oxygen generation rate, increasing the fluence rate by 189 mW/cm2 would not significantly benefit us. Microwell height and inlet flow rate involve competing phenomena—increasing height or decreasing flow reduces oxygen supply and increases PS "washout" and its concentration. [Display omitted] • The refined model for photodynamic therapy now integrates effects of ambient culture medium. • Singlet oxygen induced cellular death is expected to start from the outer regions of the spheroids. • Drug light interval has been observed to significantly influence the therapeutic outcomes. • Photosensitizers require careful selection to avoid nonuniform results. • Optimized values for various parameters have been obtained. [ABSTRACT FROM AUTHOR]

The spheroid culture system has gained significant attention as an effective in vitro model to mimic the in vivo microenvironment. Even though numerous studies were focused on developing spheroids, the structural organization of encapsulated cells within hydrogels remains a challenge. Allylated gelatin or GelAGE is used as a bioink due to its excellent physicochemical properties. In this study, GelAGE was evaluated for its capacity to induce spontaneous spheroid formation in encapsulated HepG2 cells. GelAGE was synthesized and characterized using 1HNMR spectroscopy and ninhydrin assay. Then the physicochemical and biological attributes of GelAGE hydrogel was examined. The results demonstrate that GelAGE has remarkable ability to induce the encapsulated cells to self-organize into spheroids. [ABSTRACT FROM AUTHOR]

The intricate interplay between resident cells and the extracellular matrix (ECM) profoundly influences cancer progression. In triple-negative breast cancer (TNBC), ECM architecture evolves due to the enrichment of lysyl oxidase, fibronectin, and collagen, promoting distant metastasis. Here we uncover a pivotal transcription regulatory mechanism involving the epigenetic regulator UBR7 and histone methyltransferase EZH2 in regulating transforming growth factor (TGF)-β/Smad signaling, affecting the expression of ECM genes. UBR7 loss leads to a dramatic reduction in facultative heterochromatin mark H3K27me3, activating ECM genes. UBR7 plays a crucial role in matrix deposition in adherent cancer cells and spheroids, altering collagen content and lysyl oxidase activity, directly affecting matrix stiffness and invasiveness. These findings are further validated in vivo in mice models and TNBC patients, where reduced UBR7 levels are accompanied by increased ECM component expression and activity, leading to fibrosis-mediated matrix stiffness. Thus, UBR7 is a master regulator of matrix stiffening, influencing the metastatic potential of TNBC. [Display omitted] • UBR7 is a regulator of TGF-β/SMAD signaling axis • UBR7 interacts with EZH2 to maintain H3K27me3 mark on chromatin • UBR7 in concert with EZH2 suppresses TGFB1 gene, thereby affecting ECM gene transcription • UBR7 impairs matrix stiffness, affecting the TNBC invasion in vivo Adhikari et al. demonstrate that epigenetic regulator UBR7 functions in concert with EZH2 to modulate TGF-β signaling cascade by altering H3K27me3 landscape, thereby affecting the transcription of extracellular matrix genes. Thus, UBR7 plays a key role in stiffness-mediated metastasis suppression in triple-negative breast cancer. [ABSTRACT FROM AUTHOR]

Material-assisted cartilage tissue engineering has limited application in cartilage treatment due to hypertrophic tissue formation and high cell counts required. This study aimed at investigating the potential of human mesenchymal stromal cell (hMSC) spheroids embedded in biomaterials to study the effect of biomaterial composition on cell differentiation. Pre-cultured (3 days, chondrogenic differentiation media) spheroids (250 cells/spheroid) were embedded in tyramine-modified hyaluronic acid (THA) and collagen type I (Col) composite hydrogels (four combinations of THA (12.5 vs 16.7 mg/ml) and Col (2.5 vs 1.7 mg/ml) content) at a cell density of 5 × 106 cells/ml (2 × 104 spheroids/ml). Macropellets derived from single hMSCs (2.5 × 105 cells, ScMP) or hMSC spheroids (2.5 × 105 cells, 103 spheroids, SpMP) served as control. hMSC differentiation was analyzed using glycosaminoglycan (GAG) quantification, gene expression analysis and (immuno-)histology. Embedding of hMSC spheroids in THA-Col induced chondrogenic differentiation marked by upregulation of aggrecan (ACAN) and COL2A1, and the production of GAGs. Lower THA led to more pronounced chondrogenic phenotype compared to higher THA content. Col content had no significant influence on hMSC chondrogenesis. Pellet cultures showed an upregulation in chondrogenic-associated genes and production of GAGs with less upregulation of hypertrophic-associated genes in SpMP culture compared to ScMP group. This study presents hMSC pre-culture in spheroids as promising approach to study chondrogenic differentiation after biomaterial encapsulation at low total cell count (5 × 106/ml) without compromising chondrogenic matrix production. This approach can be applied to assemble microtissues in biomaterials to generate large cartilage construct. In vitro studies investigating the chondrogenic potential of biomaterials are limited due to the low cell-cell contact of encapsulated single cells. Here, we introduce the use of pre-cultured hMSC spheroids to study chondrogenesis upon encapsulation in a biomaterial. The use of spheroids takes advantage of the high cell-cell contact within each spheroid being critical in the early chondrogenesis of hMSCs. At a low seeding density of 5·106 cells/ml (2 × 104 spheroids/ml) we demonstrated hMSC chondrogenesis and cartilaginous matrix deposition. Our results indicate that the pre-culture might have a beneficial effect on hypertrophic gene expression without compromising chondrogenic differentiation. This approach has shown potential to assemble microtissues (here spheroids) in biomaterials to generate large cartilage constructs and to study the effect of biomaterial composition on cell alignment and migration. [Display omitted] [ABSTRACT FROM AUTHOR]

A view factor is a fundamental parameter for evaluating radiative heat transfer between two surfaces. View factors for various geometries have been investigated in the past studies. However, the view factors of a spheroid and an ellipse are known only for limited configurations. In this study, the analytical view factor expressions of an ellipse and a spheroid from a plate element are derived. The spheroid view factor solution applies to a plate element with any position and orientation, and the ellipse solution is valid when the plate element is on the perpendicular axis through the ellipse center. The derived analytical solutions are validated against the numerical results for various configurations, showing deviations of less than 0.0001 for all cases. • Spheroid and ellipse view factors from a plate element are derived analytically. • The derived analytical solutions are validated against the numerical results. • Besides the general solution, simple formulas are given for specific configurations. [ABSTRACT FROM AUTHOR]

Epithelial ovarian cancer (EOC) is highly lethal due to its unique metastatic characteristics. EOC spheroids enter a non-proliferative state, with hypoxic cores and reduced oncogenic signaling, all of which contribute to tumour dormancy during metastasis. We investigated the metabolomic states of EOC cells progressing through the three steps to metastasis. Metabolomes of adherent, spheroid, and re-adherent cells were validated by isotopic metabolic flux analysis and mitochondrial functional assays to identify metabolic pathways that were previously unknown to promote EOC metastasis. Although spheroids were thought to exist in a dormant state, metabolomic analysis revealed an unexpected upregulation of energy production pathways in spheroids, accompanied by increased abundance of tricarboxylic acid (TCA) cycle and electron transport chain proteins. Tracing of 13C-labelled glucose and glutamine showed increased pyruvate carboxylation and decreased glutamine anaplerosis in spheroids. Increased reductive carboxylation suggests spheroids adjust redox homeostasis by shuttling cytosolic NADPH into mitochondria via isocitrate dehydrogenase. Indeed, we observed spheroids have increased respiratory capacity and mitochondrial ATP production. Relative to adherent cells, spheroids reduced serine consumption and metabolism, processes which were reversed upon spheroid re-adherence. The data reveal a distinct metabolism in EOC spheroids that enhances energy production by the mitochondria while maintaining a dormant state with respect to growth and proliferation. The findings advance our understanding of EOC metastasis and identify the TCA cycle and mitochondrional activity as novel targets to disrupt EOC metastasis, providing new approaches to treat advanced disease. • Epithelial ovarian cancer metastasis is driven by shedding cells that form spheroids and travel to secondary attachment sites. • Dormant, non-proliferative spheroids maintain an active metabolism, with increased TCA cycle activity. • Metabolic shifts allow spheroids to maintain adequate NADPH levels for reactive oxygen species (ROS) detoxification under hypoxic conditions. • Serine metabolism, a hallmark of carcinogenesis, is activated upon spheroid reattachment. • High metabolic potential primes spheroids for reattachment and secondary tumour formation. [ABSTRACT FROM AUTHOR]

[Display omitted] Multicellular spheroids have emerged as a robust platform to model tumor growth and are widely used for studying drug sensitivity. Diffusion is the main mechanism for transporting nutrients and chemotherapeutic drugs into spheroids, since they are typically avascular. In this study, the Bayesian inference was used to solve the inverse problem of determining the light attenuation coefficient and diffusion coefficient of Rhodamine 6G (R6G) in breast cancer spheroids, as a mock drug for the tyrosine kinase inhibitor, Neratinib. Four types of breast cancer spheroids were formed and the diffusion coefficient was estimated assuming a linear relationship between the intensity and concentration. The mathematical model used for prediction is the solution to the diffusion problem in spherical coordinates, accounting for the light attenuation. The Gaussian likelihood was used to account for the error between the measurements and model predictions. The Markov Chain Monte Carlo algorithm (MCMC) was used to sample from the posterior. The posterior predictions for the diffusion and light attenuation coefficients were provided. The results indicate that the diffusion coefficient values do not significantly vary across a HER2+ breast cancer cell line as a function of transglutaminase 2 levels, even in the presence of fibroblast cells. However, we demonstrate that different diffusion coefficient values can be ascertained from tumorigenic compared to nontumorigenic spheroids and from nonmetastatic compared to post-metastatic breast cancer cells using this approach. We also report agreement between spheroid radius, attenuation coefficient, and subsequent diffusion coefficient to give evidence of cell packing in self-assembled spheroids. The methodology presented here will allow researchers to determine diffusion in spheroids to decouple transport and drug penetration changes from biological resistivity. [ABSTRACT FROM AUTHOR]

Osteocytes play an important role to modulate the bone remodeling and are also known as terminally differentiated cells originated from the osteoblast precursor cells, but its differentiation mechanism remains unclear. Since an efficient in vitro method to evoke the osteocyte differentiation from the osteoblast precursor cells has not been established, we conducted the comparative gene expression analysis for mouse pre-osteoblast MC3T3-E1 cells in order to elucidate the key factors to induce the osteocyte differentiation from the pre-osteoblast cells. In this study, we prepared four different culture environments by modulating their cell-substrate interaction and cell–cell interaction; (i) low and (ii) high cell density on the adhesive culture models, and (iii) low and (iv) high cell density on the non-adhesive floating culture models. By comparing these conditions in terms of cell-substrate and cell–cell interaction, we showed that the elimination of cell-substrate interaction under non-adhesive floating culture greatly up-regulated the gene expression of osteocyte markers in the pre-osteoblast cells. Moreover, the presence of moderate cell–cell interaction in the non-adhesive spheroid culture further enhanced the up-regulation of osteocyte markers for the pre-osteoblast cells. The results altogether suggest the most appropriate culture environment to induce the in vitro osteocyte differentiation of pre-osteoblast cells. [ABSTRACT FROM AUTHOR]

Radiotherapy plays an essential role in the treatment of more than half of all patients with cancer. In recent decades, advances in devices that deliver radiation and the development of treatment planning software have helped radiotherapy attain precise tumour targeting with minimal toxicity to surrounding tissues. Simultaneously, as more targeted drug therapies are being brought into the market, there has been significant interest in improving cure rates for cancer by adding drugs to radiotherapy to widen the therapeutic window, the difference between normal tissue toxicity and treatment efficacy. The development of new combination therapies will require judicious adaptation of preclinical models that are routinely used for traditional drug discovery. Here we highlight the strengths and weaknesses of each of these preclinical models and discuss how they can be used optimally to identify new and clinically beneficial drug–radiotherapy combinations. [ABSTRACT FROM AUTHOR]

Hanging drop (HD) is one of the most popular methods used for forming three-dimensional (3D) cell spheroids. However, conventional hanging drop systems are only applicable for short-term spheroid culture due to their inconvenience in exchanging cell culture media. Here we present a medium-reservoir-integrated superhydrophobic (MRI-SH) chip for long-term HD spheroid cultures. The device consists of two main components: i) a patterned superhydrophobic (SH) surface containing an array of wettable spots which anchor arrays of droplets of cell suspension, and ii) an array of chambers that serve as medium reservoirs, both interconnected via an array of thru-holes. This configuration provides two distinct advantages over conventional HD configurations: i) the high wettability contrast of the SH pattern on the chip leads to the formation and adhesion of nearly spherical hanging droplets on its surface, which minimizes interactions between the liquid and the substrate; ii) the integrated chambers provide large volumes of medium to maintain longer culture durations. Using this device, spheroids of MHCC97H cells were successfully formed, and the cultured spheroids could maintain high viability for up to 30 days and exhibited enhanced spheroid morphology compared to those cultured in the conventional HD systems. This paper presents a medium-reservoir-integrated superhydrophobic hanging drop (HD) platform for the long-term culture of spheroids with enhanced morphology. By monolithically integrating medium reservoirs and a patterned SH surface into a single device, this HD platform can not only produce high-quality spheroids, but also permit them to sustain high viability for up to 30 days without the need for tedious medium replenishment. We believe that such a platform will be valuable in a wide range of biological or biomedical applications, including tissue engineering, regenerative medicine, and drug discovery. [Display omitted] [ABSTRACT FROM AUTHOR]

Three-dimensional (3D) culture systems that provide a more physiologically similar environment than conventional two-dimensional (2D) cultures have been extensively developed. Previously we have provided a facile method for the formation of 3D spheroids using non-adhesive N-hexanoyl glycol chitosan (HGC) hydrogel-coated dishes, but with limitations such as low gel stability and weak mechanical properties. In this study, chemically crosslinked hydrogels were prepared by photocrosslinking of methacrylated HGCs (M-HGCs), and their spheroid-forming abilities were evaluated for long-term 3D cell cultures. The M-HGC hydrogels demonstrated not only enhanced gel stability, but also good spheroid-forming abilities. Furthermore, the M-HGC-coated dishes were effective in generating spheroids of larger size and higher cell density depending on the crosslinking density of the M-HGCs. These results indicate that our hydrogel-coated dish system could be widely applied as an effective technique to produce cell spheroids with customized sizes and densities that are essential for tissue engineering and drug screening. Schematic illustration of 3D cell spheroid formation using non-cell-adhesive hydrogel-coated dishes. [Display omitted] • Photocrosslinkable thermogelling M-HGCs were synthesized as new non-cell adhesive hydrogels for 3D cell culture. • The photocrosslinked M-HGC hydrogels exhibited enhanced physical stability and mechanical properties. • M-HGC-coated dishes demonstrated good spheroid-forming abilities and produced large spheroids with higher cell density. • M-HGC-coated dish system could be useful to produce diverse cell spheroids with customized sizes and densities. [ABSTRACT FROM AUTHOR]

The current 2D culture model systems developed for drug screening are not sufficient to reflect the characteristics of in vivo solid tumors. Therefore, more effective in vitro tumor model systems must be developed for translational studies on therapeutic drug screening and testing. Herein, we report a new ultra-low adhesion (ULA) hydrogel for generating 3D cancer cell spheroids as tumor models in vitro. N -octanoyl glycol chitosan (OGC) was synthesized and coated onto the surface of a typical cell culture dish. Cell spheroids were effectively formed on the OGC-coated surface, and phenotypes of the tumor cells were well maintained during culture. More importantly, U373-MG cells cultured on OGC-coated plates were more resistant to doxorubicin than cells cultured on typical plates. Our OGC-based ULA system may offer a convenient method for 3D cell culture to provide enhanced performance in cancer research, drug screening and toxicology. [ABSTRACT FROM AUTHOR]

Transplantation of macroencapsulated pancreatic islets within semipermeable membranes is a promising approach for the treatment of type 1 diabetes. Encapsulation beneficially isolates the implants from the host immune system. Deleteriously however, it also limits oxygen supply to the cells. This creates challenges in loading islets at the amount and density required to meet the practical demands of clinical usage. To overcome this challenge, we investigated the feasibility of using macroporous scaffolds made of an oxygen-permeable polymer, poly(dimethylsiloxane) (PDMS) by culturing pancreatic islet-like three-dimensional tissue made of a rat pancreatic beta cell line on the scaffolds. With external oxygenation, the density and function of cells on the PDMS scaffold were more than three times and almost two times higher than those without oxygenation, respectively. This suggests that the oxygenation afforded by the PDMS scaffolds allows for high-density loading of islet tissue into the devices. [ABSTRACT FROM AUTHOR]

The majority of women with ovarian cancer are diagnosed with metastatic disease, therefore elucidating molecular events that contribute to successful metastatic dissemination may identify additional targets for therapeutic intervention and thereby positively impact survival. Using two human high grade serous ovarian cancer cell lines with inactive TP53 and multiple rounds of serial in vivo passaging, we generated sublines with significantly accelerated intra-peritoneal (IP) growth. Comparative analysis of the parental and IP sublines identified a common panel of differentially expressed genes. The most highly differentially expressed gene, upregulated by 60-65-fold in IP-selected sublines, was the type I transmembrane protein AMIGO2. As the role of AMIGO2 in ovarian cancer metastasis remains unexplored, CRISPR/Cas9 was used to reduce AMIGO2 expression, followed by in vitro and in vivo functional analyses. Knockdown of AMIGO2 modified the sphere-forming potential of ovarian cancer cells, reduced adhesion and invasion in vitro, and significantly attenuated IP metastasis. These data highlight AMIGO2 as a new target for a novel anti-metastatic therapeutic approach aimed at blocking cohesion, survival, and adhesion of metastatic tumorspheres. [ABSTRACT FROM AUTHOR]

• Development of a microfluidic system to evaluate candidate drugs with clinical radiotherapy on tumor spheroids. • The orthovoltage-based irradiation technique enabled the delivery of four different radiation doses on one microfluidic system. • A synergistic effect of Talazoparib with radiotherapy on soft tissue sarcoma spheroids was demonstrated with clonogenic assays. • This tool lays the basis for the systemic search of molecular agent/radiotherapy synergies. Radioresistance, tumor microenvironment, and normal tissue toxicity from radiation limit the efficacy of radiotherapy in treating cancers. These challenges can be tackled by the discovery of new radiosensitizing and radioprotecting agents aimed at increasing the therapeutic efficacy of radiotherapy. The goal of this work was to develop a miniaturized microfluidic platform for the discovery of drugs that could be used in combination with radiotherapy. The microfluidic system will allow the toxicity testing of cancer spheroids to different combinations of radiotherapy and molecular agents. An orthovoltage-based technique was used to expose the devices to multiple X-ray radiation doses simultaneously. Radiation dose-dependent DNA double-strand breaks in soft tissue sarcoma (STS) spheroids were quantified using comet assays. Analysis of proliferative death using clonogenic assays was also performed, and synergy between treatments with Talazoparib, Pazopanib, AZD7762, and radiotherapy was quantified using dedicated statistical tests. The developed microfluidic system with simple magnetic valves was capable of growing 336 homogeneous STS spheroids. The irradiation of the microfluidic system with an orthovoltage-based technique enabled the screening of sixteen drug-radiotherapy combinations with minimal reagent consumption. Using this framework, we predicted a therapeutic synergy between a novel anticancer drug Talazoparib and radiotherapy for STS. No synergy was found between RT and either Pazopanib or AZD7762, as the combinations were found to be additive. This methodology lays the basis for the systemic search for molecular agent/radiotherapy synergies among preexisting pharmaceutical compounds libraries, in the hope to identify failed drug candidates in monotherapy that, in the presence of radiotherapy, would make it through clinical trials. [ABSTRACT FROM AUTHOR]

The placenta is a unique organ with an active metabolism and dynamically changing physiology throughout pregnancy. It is difficult to elucidate the structure of cell-cell and cell-extracellular matrix interactions of the placenta in in vivo studies due to interspecies differences and ethical constraints. In this study, human umbilical cord vein cells (HUVEC) and human placental choriocarcinoma cells (BeWo) were co-cultured for the first time to form spheroids (microtissues) on a three-dimensional (3D) Petri Dish® mold and compared with a traditional two-dimensional (2D) system. Vortioxetine is an antidepressant with a lack of literature on its use in pregnancy in established cultures, the toxicity of vortioxetine was studied to investigate the response of spheroids representing placental tissue. Spheroids were characterised by morphology and exposed to vortioxetine. Cell viability and barrier integrity were then measured. Intercellular junctions and the localisation of serotonin transporter (SERT) proteins were demonstrated by immunofluorescence (IF) staining in BeWo cells. Human chorionic gonadotropin (beta-hCG) hormone levels were also measured. In the 3D system, cell viability and hormone production were higher than in the 2D system. It was observed that the barrier structure was impaired, the structure of intracellular skeletal elements was altered and SERT expression decreased depending on vortioxetine exposure. These results demonstrate that the multicellular microtissue placenta model can be used to obtain results that more closely resemble in vivo toxicity studies of various xenobiotics than other 2D and mono-culture spheroid models in the literature. It also describes the use of 3D models for soft tissues other than the placenta. [Display omitted] • BeWo and HUVEC microtissues formed in 3D Petri Dish® for the first time. • A novel placental tissue model was established. • Vortioxetine treatment disrupted microtissue integrity. • Cells appeared to be more resistant in co-cultured microtissues. • β-hCG hormone levels were higher in 3D systems than in 2D systems. [ABSTRACT FROM AUTHOR]

Curcumin exhibits a broad spectrum of beneficial health properties that include anti-tumor and anti-cancer activities. The down-regulation of c-myc transcription via stabilizing the G-quadruplex structure formed at the promoter region of the human c-myc gene allows the repression in cancer growth. Small molecules can bind and stabilize this structure to provide an exciting and promising strategy for anti-cancer therapeutics. Herein, we investigated the interaction of Curcumin and its synthetic analogs with G-quadruplex DNA formed at the c-myc promoter by using various biophysical and biochemical assays. Further, its cytotoxic effect and mechanistic insights were explored in various cancer cell lines as well as in multicellular tumor spheroid (MCTS) model. The MCTS possesses almost similar microenvironment as avascular tumors, and micro-metastases can be used as a suitable model for the small molecule-based therapeutics development. Our study provides an expanded overview of the anti-cancer effect of a new Curcumin analog via targeting G-quadruplex structures formed at the promoter region of the human c-myc gene. Image 1 • The Curcumin analogs showed promising scaffold to interact with c-myc G-quadruplex DNA. • The lead molecule Cur-4 exhibited strong binding affinity and stability for c-myc G-quadruplex. • Cur-4 significantly reduced the c-myc gene transcription and expression in HeLa cells. • Cur-4 also uniformly distributed in the three-dimensional tumor spheroid of HeLa cells. [ABSTRACT FROM AUTHOR]

Efficacy of chemotherapy is limited by the resistance of cancer cells. Phytochemicals especially Essential Oils (EOs) provide an alternative mode of cancer therapy. However, EOs utilization is restricted because of low bioavailability, and high degradation. Nanoemulsification is a method developed to overcome these obstacles. Accordingly, Citrus-Pectin nanoemulsion of Zataria Essential Oil (CP/ZEONE) was prepared to evaluate the anticancer activity and the mechanisms responsible for the caused cytotoxicity. Physical properties and FTIR spectra of CP/ZEONE were characterized. CP/ZEONE progressively improves the suppression of viability of drug-resistant MCF-7, MDA-MB-231 breast cancer cells, and spheroids. It triggers apoptosis by increasing Reactive Oxygen Species (ROS), mitochondrial membrane potential (MMP) loss, DNA damage, G2 and S-phase arrest in MDA-MB-231 cells and spheroids respectively. Additionally, spectroscopy techniques revealed the interaction of CP/ZEONE with DNA via the formation of a groove binding/partial intercalative complex. Thus, ZEO-loaded CP Nano-particles can be further explored as a promising antiproliferative and therapeutic candidate against cancer. [ABSTRACT FROM AUTHOR]

The acoustic spectrum of a gas-filled resonating cavity can be used to indirectly probe its internal velocity field. This unconventional velocimetry method is particularly interesting for opaque fluid or rapidly rotating flows, which cannot be imaged with standard methods. This requires to (i) identify a large enough number of acoustic modes, (ii) accurately measure their frequencies, and (iii) compare with theoretical synthetic spectra. Relying on a dedicated experiment, an air-filled rotating spheroid of moderate ellipticity, our study addresses these three challenges. To do so, we use a comprehensive theoretical framework, together with finite-element calculations, and consider symmetry arguments. We show that the effects of the Coriolis force can be successfully retrieved through our acoustic measurements, providing the first experimental measurements of the rotational splitting (or Ledoux) coefficients for a large collection of modes. Our results pave the way for the modal acoustic velocimetry to be a robust, versatile, and non-intrusive method for mapping large-scale flows. [ABSTRACT FROM AUTHOR]

Les avancées sur le plan physique, technologique et biologique ont fait de l'oncologie radiothérapie une discipline en constante progression. De nouveaux axes de recherche actuels pourraient être transférés en clinique dans un favenir proche. Dans cette revue, sous la forme d'interviews, différentes thématiques prometteuses pour notre spécialité sont décrites, telles que le microbiote intestinal, les organoïdes tumoraux (ou avatar), l'intelligence artificielle, les thérapies connectées, les nanotechnologies et le laser plasma. La prédiction individuelle du meilleur index thérapeutique combinée à l'intégration de nouvelles technologies permettra idéalement le traitement hautement personnalisé des patients pris en charge en oncologie radiothérapie. Advances in physical, technological and biological fields have made radiation oncology a discipline in continual evolution. New current research areas could be implemented in the clinic in the near future. In this review in the form of several interviews, various promising themes for our specialty are described such as the gut microbiota, tumor organoids (or avatar), artificial intelligence, connected therapies, nanotechnologies and plasma laser. The individual prediction of the best therapeutic index combined with the integration of new technologies will ideally allow highly personalized treatment of patients receiving radiation therapy. [ABSTRACT FROM AUTHOR]

• We found the thermoelectric field distribution of spheroid inside a matrix material • The Maxwell–Garnett approach was adopted for granular thermoelectric materials • The model was illustrated for MgAg 0.97 Sb 0.99 inclusions placed in a matrix of Bi 2 Te 3 • Impact of the spheroid geometry on effective properties of a composite is analyzed The paper studies the problem of an extended thermoelectric spheroid placed in a thermoelectric matrix. The external electric field is assumed to be uniform. Despite the nonlinear nature of the equations, the solution of the problem can be found as a sum of Legendre functions and polynomials. The solution obtained is used to find the effective values of electrical and thermal conductivities, as well as the thermopower for a composite medium consisting of a set of randomly located spheroids. As an example of the model application, we consider a composite consisting of spheroidal inclusions MgAg 0.97 Sb 0.99 placed in a matrix of Bi 2 Te 3. [ABSTRACT FROM AUTHOR]

Simulations of flows containing non-spherical particles (fibres or ellipsoids) rely on the knowledge of the equation governing the particle motion in the flow. Most models used nowadays are based on the pioneering work of Jeffery (1922), who obtained an equation for the motion of an ellipsoidal particle immersed in a Newtonian fluid, despite the fact that this model relies on strong assumptions: negligible inertia, unconfined flow, dilute regime, flow unperturbed by the presence of the suspended particle, etc. In this work, we propose a dumbbell-based model aimed to describe the motion of an inertial fibre or ellipsoid suspended in a Newtonian fluid. We then use this model to study the orientation kinematics of such particle in a linear shear flow and compare it to the inertialess case. In the case of fibres, we observe the appearance of periodic orbits (whereas inertialess fibres just align in the flow field). For spheroids, our model predicts an orbit drift towards the flow-gradient plane, either gradually (slight inertia) or by first rotating around a moving oblique axis (heavy particles). Multi-Particle Collision Dynamics (MPCD) simulations were carried out to assess the model predictions in the case of inertial fibres and revealed similar behaviours. [ABSTRACT FROM AUTHOR]

Cyanobacteria are known to produce many toxins and other secondary metabolites. The study of their specific mode of action may reveal the biotechnological potential of such compounds. Portoamides A and B (PAB) are cyclic peptides isolated from the cyanobacteria Phormidium sp. due to their growth repression effect on microalgae and were shown to be cytotoxic against certain cancer cell lines. In the present work, viability was assessed on HCT116 colon cancer cells grown as monolayer culture and as multicellular spheroids (MTS), non-carcinogenic cells and on zebrafish larvae. HCT116 cells and epithelial RPE-1hTERT cells showed very similar degrees of sensitivities to PAB. PAB were able to penetrate the MTS, showing a four-fold high IC 50 compared to monolayer cultures. The toxicity of PAB was similar at 4 °C and 37 °C suggesting energy-independent uptake. PAB exposure decreased ATP production, mitochondrial maximal respiration rates and induced mitochondrial membrane hyperpolarization. PAB induced general organelle stress response, indicated by an increase of the mitochondrial damage sensor PINK-1, and of phosphorylation of eIF2α, characteristic for endoplasmic reticulum stress. In summary, these findings show general toxicity of PAB on immortalized cells, cancer cells and zebrafish embryos, likely due to mitochondrial toxicity. Image 1 • Colon carcinoma cells and normal epithelial cells demonstrated similar sensitivity to portoamides. • Zebrafish larvae were strongly affected by portoamides. • PAB had prominent cytotoxic effects on the prolific outer layer of spheroids. • Toxicity of PAB is suggested via energy-independent uptake. • PAB induced mitochondrial toxicity (ATP production, respiration rates, mitochondrial membrane hyperpolarization). [ABSTRACT FROM AUTHOR]

Bilirubin is excreted into the bile from hepatocytes, mainly as monoglucuronosyl and bisglucuronosyl conjugates, reflecting bilirubin glucuronidation activity. However, there is limited information on the in vitro evaluation of liver cell lines or primary hepatocytes. This study aimed to investigate variations in the bilirubin metabolic function of canine and human hepatocyte spheroids formed in a three-dimensional (3D) culture system indicated by the formation of bilirubin glucuronides when protease inhibitors such as atazanavir, indinavir, ritonavir, and nelfinavir were treated with bilirubin. The culture supernatant was collected for bilirubin glucuronidation assessment and the cells were used to evaluate viability. On day 8 of culture, both canine and human hepatocyte spheroids showed high albumin secretion and distinct spheroid formation, and their bilirubin glucuronidation activities were evaluated considering cell viability. Treatment with atazanavir and ritonavir remarkably inhibited bilirubin glucuronide formation, wherein atazanavir showed the highest inhibition, particularly in human hepatocyte spheroids. These results may reflect the effects on cellular uptake of bilirubin and its intracellular metabolic function. Thus, primary hepatocytes cultured in a 3D culture system may be a useful in vitro system for the comprehensive evaluation of bilirubin metabolic function and risk assessment in bilirubin metabolic disorders for drug development. • Hepatocyte spheroids cultured in a 3D system indicate bilirubin metabolic function. • Canine and human hepatocyte spheroids showed high albumin secretion. • Human hepatocyte spheroids showed higher UGT1A1 expression than that in canines. • Albumin secretion by canine hepatocyte spheroids in a 3D system is a novel finding. • Culturing primary hepatocytes in 3D systems have implications in drug development. [ABSTRACT FROM AUTHOR]

Adequate invasion and complete remodelling of the maternal spiral arteries by the invading extravillous trophoblasts are the major determinants of a successful pregnancy. Increase in oxidative stress during pregnancy has been linked to the reduction in trophoblast invasion and incomplete conversion of the maternal spiral arteries, resulting in pregnancy complications such as pre-eclampsia, intrauterine growth restriction, and spontaneous miscarriages resulting in foetal/maternal mortality. The use of antioxidant therapy (vitamin C and E) and other preventative treatments (such as low dose aspirin) have been ineffective in preventing pre-eclampsia. Also, as the majority of antihypertensive drugs pose side effects, choosing an appropriate treatment would depend upon the efficacy and safety of mother/foetus. Since pre-eclampsia is mainly linked to placental oxidative stress, new diet-based antioxidants can be of use to prevent this condition. The antioxidant properties of flavonoids (naturally occurring phenolic compounds which are ubiquitously distributed in fruits and vegetables) have been well documented in non-trophoblast cells. Therefore, this study aimed to investigate the effects of flavonoids (quercetin, hesperidin) and their metabolites (Quercetin 3-O-β-glucuronide and hesperetin), either alone or in combination, on first trimester trophoblast cell line HTR-8/SVneo during oxidative stress. The data obtained from this study indicate that selected flavonoids, their respective metabolites significantly reduced the levels of reduced glutathione (p < 0.0001) during HR-induced oxidative stress. These flavonoids also inhibited the activation of pro-apoptotic kinases (p38 MAPK and c-Jun N-terminal kinase) during HR-induced phosphorylation. In addition, they enhanced spheroid stem-like cell generation from HTR8/SVneo cells, aiding their invasion. Our data suggest that dietary intake of food rich in quercetin or hesperidin during early pregnancy can significantly improve trophoblast (placenta) health and function against oxidative stress. [ABSTRACT FROM AUTHOR]

Inertial migration of single neutrally buoyant prolate and oblate rigid spheroids in a plane Poiseuille flow at Reynolds number Re ≈ 100 – 500 is studied using dissipative particle dynamics (DPD) simulations. Inertial migration of single neutrally buoyant prolate and oblate rigid spheroids in a plane Poiseuille flow at Reynolds number is studied using dissipative particle dynamics (DPD) simulations. The particles have aspect ratios ( AR p , ratio of the major axis L to the minor axis B) from unity to three for prolate and from unity to eight for oblate spheroids, and the ratio of the diameter of the major axis to the gap (L / H) ranges from 0.2 to around 0.4 for both types of the spheroids. The equilibrium positions move closer to the channel center as Re , AR p , and L / H increase. The previously observed effect of the maximum blockage ratio L / H on the equilibrium position is re-confirmed, while differences for oblate vs. prolate shape at fixed L / H , deemed negligible by previous studies, are found in our study to be significant for both the equilibrium position and the rotational behavior, at L / H ⩾ 0.3 and Re ⩾ 200. The major axis of a prolate spheroid generally tumbles in the flow-gradient plane, and the dependence of its focusing position on L / H likely arises from interactions with the wall during tumbling. We observed that when Re is above around 300, with increasing AR p , an oblate spheroid changes from log-rolling with the minor axis in the vorticity direction ( AR p = 1.5), to inclined-rolling with the minor axis tilted at an angle with respect to the vorticity axis ( AR p = 3), to a mixed state of inclined-rolling and inclined-tumbling ( AR p = 5), and finally to an approximate steady state, where the minor axis remains in the flow-gradient plane and slowly fluctuates between 60 ° and 90 ° from the flow direction ( AR p = 8). The utility of the DPD methods for exploring inertial migration phenomena of particles of various shapes is thus demonstrated. [ABSTRACT FROM AUTHOR]

Abstract Interconnected macroporous hydrogel is hydrophilic; it exhibits soft tissue-like mechanical property and aqueous-stable macroporosity for 3D spheroid culture. There is an unmet need to develop cleavable macroporous hydrogel, for the ease of retrieving functional spheroids for further in vitro and in vivo applications. We have developed and comprehensively characterized a hydroxypropyl-cellulose-disulfide sponge by systematically identifying strategies and synthesis schemes to confer cleavability to the sponge under cell-friendly conditions. It preserved the essential advantages of the macroporous hydrogel to support 3D spheroid formation and maintenance of sensitive hepatocytes while allowing rapid cleavage and retrieval of functional spheroids. By culturing HepaRG as spheroids in the cleavable sponge, we have accelerated HepaRG differentiation to 9 days compared to 28 days in 2D culture. Cytochrome P450 basal activity reached significantly higher level, while albumin secretion and fluorescein diacetate staining indicated the same at day 5. The purity of albumin+ hepatocytes reached 92.9% versus 7.1% of CK19+ cholangiocytes at day 9, a much stronger preference for hepatocytes than the 60% albumin+ hepatocytes purity in 2D culture. HepaRG differentiated hepatocytes were retrieved by cleaving the sponge with 10 mM tris-(2-carboxyethyl)-phosphine (TCEP) within 30 min preserving viability, plateability and positive albumin staining of the hepatocyte spheroids. This cleavable macroporous hydrogel sponge will support the rapid development of various 3D spheroid- or organoid-based applications in basic research and drug testing. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Elastin-like polypeptide (ELP) nanoparticles are a versatile platform for targeted drug delivery. As for any type of nanocarrier system, an important challenge remains the ability of deep (tumor) tissue penetration. In this study, ELP particles with controlled surface density of the cell-penetrating peptide (CPP) octa-arginine (R8) were created by temperature-induced co-assembly. ELPs formed micellar nanoparticles with a diameter of around 60 nm. Cellular uptake in human skin fibroblasts was directly dependent on the surface density of R8 as confirmed by flow cytometry and confocal laser scanning microscopy. Remarkably, next to promoting cellular uptake, the presence of the CPP also enhanced penetration into spheroids generated from human glioblastoma U-87 cells. After 24 h, uptake into cells was observed in multiple layers towards the spheroid core. ELP particles not carrying any CPP did not penetrate. Clearly, a high CPP density exerted a dual benefit on cellular uptake and tissue penetration. At low nanoparticle concentration, there was evidence of a binding site barrier as observed for the penetration of molecules binding with high affinity to cell surface receptors. In conclusion, R8-functionalized ELP nanoparticles form an excellent delivery vehicle that combines tunability of surface characteristics with small and well-defined size. [ABSTRACT FROM AUTHOR]

Abstract Successful tissue engineering requires the generation of human scale implants that mimic the structure, composition and mechanical properties of native tissues. Here, we report a novel biofabrication strategy that enables the engineering of structurally organised tissues by guiding the growth of cellular spheroids within arrays of 3D printed polymeric microchambers. With the goal of engineering stratified articular cartilage, inkjet bioprinting was used to deposit defined numbers of mesenchymal stromal cells (MSCs) and chondrocytes into pre-printed microchambers. These jetted cell suspensions rapidly underwent condensation within the hydrophobic microchambers, leading to the formation of organised arrays of cellular spheroids. The microchambers were also designed to provide boundary conditions to these spheroids, guiding their growth and eventual fusion, leading to the development of stratified cartilage tissue with a depth-dependant collagen fiber architecture that mimicked the structure of native articular cartilage. Furthermore, the composition and biomechanical properties of the bioprinted cartilage was also comparable to the native tissue. Using multi-tool biofabrication, we were also able to engineer anatomically accurate, human scale, osteochondral templates by printing this microchamber system on top of a hypertrophic cartilage region designed to support endochondral bone formation and then maintaining the entire construct in long-term bioreactor culture to enhance tissue development. This bioprinting strategy provides a versatile and scalable approach to engineer structurally organised cartilage tissues for joint resurfacing applications. [ABSTRACT FROM AUTHOR]

Abstract Perfluorooctanoic acid (PFOA) toxicity is of considerable concern due to its wide application, environmental persistence, and bioaccumulation. In the current study, we used a scaffold-free three-dimensional (3D) spheroid model of mouse liver cells (AML12) to explore the toxicity of PFOA and emerging alternatives (HFPO-DA and PFO4DA). Comparing the short-term (24 and 72 h treatment) toxicity of PFOA between conventional 2D monolayer cells and 3D spheroids, we found that spheroids had higher EC 50 values and lower ROS levels after treatment, indicating their greater resistance to PFOA. Cell viability (i.e., adenosine triphosphate (ATP) content and lactate dehydrogenase (LDH) leakage) and liver-specific function (i.e., albumin secretion) were stable in spheroids through 28 day of culture. However, under 100 and 200 μM-PFOA treatment for 28 day, ROS levels, LDH leakage, and caspase3/7 activity all increased significantly. As a sensitive parameter, ROS showed a significant increase at 21 day, even in the 50 μM-PFOA group. Consistent with the elevation of ROS and caspase3/7, the expressions of oxidative stress- and apoptosis-related genes, including Gsta2 , Nqo1 , Ho-1 , caspase3 , p53 , and p21 , were induced in dose- and time-dependent manners after PFOA exposure. The peroxisome proliferator-activated receptor alpha (PPARα) pathway was also activated after treatment, with significant induction of its target genes, Fabp4 and Scd1. Similar to PFOA, both HFPO-DA and PFO4DA activated the PPARα pathway, induced ROS levels, and initiated cell damage, though at a relatively lower extent than that of PFOA. Our results imply that the 3D spheroid model is a valuable tool in chronic toxicological studies. Graphical abstract Image 1 Highlights • PFOA toxicity differed between the 3D spheroid model and 2D monolayer cells. • ROS was a sensitive PFOA parameter in spheroids after long-term exposure. • PFOA activated the PPARα pathway in spheroids after long-term exposure. • HFPO-DA and PFO4DA hepatotoxicities were lower than that of PFOA. • The spheroid model is a feasible in vitro approach in chronic toxicological studies. [ABSTRACT FROM AUTHOR]

Abstract In recent years, in vitro three-dimensional (3D) cell culture systems that better mimic in vivo physiology and tissue microenvironment have become important. It is used for the pre-clinical evaluation of the efficacy and safety profiles of new drug candidates developed especially for the treatment of various diseases including cancer. 3D cell culture techniques overcome the inadequacy of conventional two-dimensional (2D) cell culture models as they are much more informative than the 2D systems. And they can easily replace or reduce the number of in vivo studies which arise many ethical problems in the development of novel therapeutics. This review focuses on the organoid cultures which are 3D bioengineered cell culture systems that guide cell behavior and cell order to mimic in vivo tumor microenvironment. Graphical abstract Unlabelled Image Highlights • Person specific organoids mimic in vivo system better than other 3D tissue models. • Organoids are modelled for several applications such as cancer by using stem cells. • Application of organoid culture with CRISPR and microfluidic technology is novel. [ABSTRACT FROM AUTHOR]

Abstract To carry out numerical simulations on the particle-laden supercritical water (SCW), one needs to know drag coefficient (Cd) and average Nusselt number (Nu) of the solid particles in SCW according to the high temperature and pressure conditions. This study conducts particle-resolved direct numerical simulations (PR-DNS) to obtain Cd and Nu of spheroids in SCW whose physical properties significantly change with temperature and pressure. A series of case studies are designed to match different working conditions. Effects of variable physical property parameters of SCW on Cd and Nu of spheroids are discussed. Numerical results show that the variable viscosity of SCW plays a more important role in influencing on Cd of spheroids in SCW rather than density, thermal conductivity or specific heat capacity. All these four parameters could affect Nu and the largest effect comes from the specific heat capacity. New correlations of Cd and Nu for spheroids in SCW are proposed according to the numerical results which can facilitate the phase coupling in multi-phase flow modellings of SCW. Graphical abstract Unlabelled Image Highlights • Momentum and heat transfer of spheroids in SCW are studied. • Effects of SCW properties, particle shape and Reynolds number are discussed. • Correlations for Cd and Nu of spheroids in SCW are established. [ABSTRACT FROM AUTHOR]

Abstract The maintenance and direction of stem cell lineage after implantation remains challenging for clinical translation. Aggregation and encapsulation into instructive biomaterials after preconditioning can bolster retention of differentiated phenotypes. Since these procedures do not depend on cell type or lineage, we hypothesized we could use a common, tunable platform to engineer formulations that retain and enhance multiple lineages from different cell populations. To test this, we varied alginate stiffness and adhesive ligand content, then encapsulated spheroids of varying cellularity. We used Design-of-Experiments to determine the effect of these parameters and their interactions on phenotype retention. The combination of parameters leading to maximal differentiation varied with lineage and cell type, inducing a 2–4-fold increase over non-optimized levels. Phenotype was also retained for 4 weeks in a murine subcutaneous model. This widely applicable approach can facilitate translation of cell-based therapies by instructing phenotype in situ without prolonged induction or costly growth factors. [ABSTRACT FROM AUTHOR]

Abstract Multicellular spheroids provide a physiologically relevant platform to study the microenvironment of tumors and therapeutic applications, such as microparticle-based drug delivery. The goal of this study was to investigate the incorporation/penetration of compliant polyacrylamide microparticles (MPs), into either cancer or normal human cell spheroids. Incorporation of collagen-1-coated MPs (stiffness: 0.1 and 9 kPa; diameter: 15–30 µm) into spheroids (diameter ∼100 µm) was tracked for up to 22 h. Results indicated that cells within melanoma spheroids were more influenced by MP mechanical properties than cells within normal cell spheroids. Melanoma spheroids had a greater propensity to incorporate and displace the more compliant MPs over time. Mature spheroids composed of either cell type were able to recognize and integrate MPs. While many tumor models exist to study drug delivery and efficacy, the study of uptake and incorporation of cell-sized MPs into established spheroids/tissues or tumors has been limited. The ability of hyper-compliant MPs to successfully penetrate 3D tumor models with natural extracellular matrix deposition provides a novel platform for potential delivery of drugs and other therapeutics into the core of tumors and micrometastases. [ABSTRACT FROM AUTHOR]

Graphical abstract Highlights • A reversibly sealed microfluidic platform for 3D tumor microtissue formation and analysis is developed. • After the assay, the resealable chip allows selective spheroid retrieval for in-depth analysis. • On and off-chip characterization of 3D microtissue is carried out. • Cytotoxicity effect of drug was analyzed by using three different methods (optical, fluorescence and scanning electron microscopy). • We analyzed the SEM images to study the morphology parameters of spheroids subjected to drug treatments. Abstract Nowadays, microfluidic 3D cell culture is widely used to mimic complex microtissue and dynamic environment, performing more realistic in vitro assays for drug testing. Herein, we developed a novel microfluidic platform for tumor microtissue culture, drug response analysis and versatile microscopic characterization. By reversibly bonding the chip, we go beyond the on/off chip tradeoff, which allows us to perform both fluorescence and SEM characterization of tumor microtissues on a simple platform. The microfluidic chip consists of spherical microwells connected via microchannels, bonded through a magnetic system. Colorectal cancer HT-29 cells were cultured as spherical microtissues on chip and their growth kinetics monitored. The cytotoxic activity of Camptothecin was evaluated by in situ live/dead fluorescence staining and quantification of morphology parameters. Finally, we demonstrated the possibility to collect the 3D tumor microtissues and characterize their surface damaged by the drug using scanning electron microscopy. This reversibly sealed microfluidic platform thus enables to grow sets of 3D tumor microtissues in a controlled dynamic microenvinroment, and subsequently to retrieve the 3D tumor microtissues after chemotherapeutic treatment for in-depth analysis. [ABSTRACT FROM AUTHOR]

Abstract The multiple cavity model of nanoporous solid has been developed which takes into account the elastic stiffness of matrix solid, porosity, shape, size and arrangement of cavities as well as the effects of their interaction. To describe the pertinent to nano level size effect of the stress field and effective stiffness, the Gurtin–Murdoch model that includes both surface tension and surface stiffness has been applied. A complete semi-analytical solution to the model problem has been obtained by the multipole expansion method. The displacement perturbation field induced by each cavity is expanded over a set of the vector solutions of Lamé equation in spheroidal basis. Expansion of the displacement and stress fields in terms of the vector surface harmonics reduces the surface conditions to an infinite linear system for the multipole strengths. The superposition principle and re-expansion formulas for the partial vector solutions extend this theory to problems involving multiple cavities. The method provides accurate evaluation of the stress field with the interaction effects taken into account. Also, the derived solution constitutes a mathematical background of the modified Maxwell homogenization scheme. The stiffness tensor of nanoporous solid is found by equating the induced dipole moment of equivalent inclusion to the total dipole moment of individual cavities. Numerical study demonstrate an accuracy and computational efficiency of the developed approach and shows quite a significant combined effect of the cavity shape and surface tension and stiffness on the stress field and macroscopic stiffness of nanoporous solid. [ABSTRACT FROM AUTHOR]

Graphical abstract Abstract Photochemical internalisation (PCI) is a method for enhancing delivery of drugs to their intracellular target sites of action. In this study we investigated the efficacy of PCI using a porphyrin photosensitiser and a cytotoxic agent on spheroid and non-spheroid compressed collagen 3D constructs of ovarian cancer versus conventional 2D culture. The therapeutic responses of two human carcinoma cell lines (SKOV3 and HEY) were compared using a range of assays including optical imaging. The treatment was shown to be effective in non-spheroid constructs of both cell lines causing a significant and synergistic reduction in cell viability measured at 48 or 96 h post-illumination. In the larger spheroid constructs, PCI was still effective but required higher saporin and photosensitiser doses. Moreover, in contrast to the 2D and non-spheroid experiments, where comparable efficacy was found for the two cell lines, HEY spheroid constructs were found to be more susceptible to PCI and a lower dose of saporin could be used. PCI treatment was observed to induce death principally by apoptosis in the 3D constructs compared to the mostly necrotic cell death caused by PDT. At low oxygen levels (1%) both PDT and PCI were significantly less effective in the constructs. Statement of significance Assessment of new drugs or delivery systems for cancer therapy prior to conducting in vivo studies often relies on the use of conventional 2D cell culture, however 3D cancer constructs can provide more physiologically relevant information owing to their 3D architecture and the presence of an extracellular matrix. This study investigates the efficacy of Photochemical Internalisation mediated drug delivery in 3D constructs. In 3D cultures, both oxygen and drug delivery to the cells are limited by diffusion through the extracellular matrix unlike 2D models, and in our model we have used compressed collagen constructs where the density of collagen mimics physiological values. These 3D constructs are therefore well suited to studying drug delivery using PCI. Our study highlights the potential of these constructs for identifying differences in therapeutic response to PCI of two ovarian carcinoma lines. [ABSTRACT FROM AUTHOR]

In this study, an experimental and a numerical investigations are performed to investigate the effect of particle’s shape and size distribution on its dispersion behavior. Firstly, particle dispersion of pulverized coal and spherical polymer particles is observed by Particle Image Velocimetry (PIV) technique in the experiment. Secondly, a simulation is performed to analyze the particle dispersion in detail. Spherical and spheroidal motion models are applied to particle’s movement to investigate the shape effect. Furthermore, monodisperse and polydisperse for particles are applied to investigate the size distribution effect on the dispersion. Experimental results show that in the jet turbulence flow, pulverized coal particles, which have complex shapes and various sizes, have quite different dispersion behavior compared to spherical particles. In terms of the results of the simulation, this difference is mainly caused by the size distribution effect. Although particle’s shape affects the dispersity, it is weakened by the size distribution effect. [ABSTRACT FROM AUTHOR]

Abstract NIMA Related Kinase 6 (Nek6) is a protein kinase involved in various cellular processes, including cell cycle regulation, apopotosis, senescence, telomere maintainance and chemoresistance. In the present study, we investigated the role of Nek6 in breast tumorigenesis and the prognostic merit of Nek6 expression in breast cancer. Immunohistochemistry and Western blot analyses was conducted to determine the expression profile of Nek6 in 133 breast cancer specimens. Nek6 was overexpressed in a majority of breast cancer specimens, compared with the adjacent non-tumorous tissues. Furthermore, we revealed that high expression of Nek6 was associated with histologic grade, tumor size and TNM stage in breast cancer. Liner regression analysis showed a significant association between the levels of Nek6 and Ki-67. Cox regression analysis indicated that Nek6 expression was an independent prognostic predictor for breast cancer. Moreover, intracellular level of Nek6 was remarkably increased following the release from serum starvation in MCF-7 cells. EdU incorporation assay indicated that depletion of Nek6 remarkably impaired the proliferation of MCF-7 cells. Finally, spheroid formation assay revealed that interference of Nek6 led to diminished oncospheroid-forming capacity of breast cancer cells. In conclusion, our results imply that Nek6 plays a facilitating role in breast cancer cell proliferation and may serve as a promising therapeutic target for breast cancer. [ABSTRACT FROM AUTHOR]