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A new channel‐free water‐in‐oil (WO) droplet 3D cell culture method is proposed to address the challenges while maintaining the advantages of the conventional 3D cell culture methods. The proposed WO method can fundamentally solve the constraint of spheroids size, a common challenge in conventional 3D culture, by using droplet size controllability. The 3D cell culture performance of the WO method is verified by comparing it with the conventional 3D cell culture methods. A systematic investigation of the culture conditions of the WO method confirms the working range of cell concentration and droplet size, as well as the scalability of spheroid size. Adjusting droplet size and cell concentration enables rapid spheroid formation with large and high cell concentration droplets or fast spheroid growth with small and low cell concentration droplets, providing control over the spheroid size and growth rate according to the purpose. Furthermore, long‐term culture is demonstrated for 1 month with the proposed method, showing the largest spheroid culture and demonstrating the possibility that this method can be used not only for spheroid formation but also for organoid studies. Finally, if a WO‐based automated 3D cell culture system is developed, it will be a useful tool for organoid research. [ABSTRACT FROM AUTHOR]

Background: Synovial sarcoma (SS) is an SS18‐SSX fusion gene‐driven soft tissue sarcoma with mesenchymal characteristics, associated with a poor prognosis due to frequent metastasis to a distant organ, such as the lung. Histone deacetylase (HDAC) inhibitors (HDACis) are arising as potent molecular targeted drugs, as HDACi treatment disrupts the SS oncoprotein complex, which includes HDACs, in addition to general HDACi effects. To provide further molecular evidence for the advantages of HDACi treatment and its limitations due to drug resistance induced by the microenvironment in SS cells, we examined cellular responses to HDACi treatment in combination with two‐dimensional (2D) and 3D culture conditions. Methods: Using several SS cell lines, biochemical and cell biological assays were performed with romidepsin, an HDAC1/2 selective inhibitor. SN38 was concomitantly used as an ameliorant drug with romidepsin treatment. Cytostasis, apoptosis induction, and MHC class I polypeptide‐related sequence A/B (MICA/B) induction were monitored to evaluate the drug efficacy. In addition to the conventional 2D culture condition, spheroid culture was adopted to evaluate the influence of cell‐mass microenvironment on chemoresistance. Results: By monitoring the cellular behavior with romidepsin and/or SN38 in SS cells, we observed that responsiveness is diverse in each cell line. In the apoptotic inducible cells, co‐treatment with SN38 enhanced cell death. In nonapoptotic inducible cells, cytostasis and MICA/B induction were observed, and SN38 improved MICA/B induction further. As a novel efficacy of SN38, we revealed TWIST1 suppression in SS cells. In the spheroid (3D) condition, romidepsin efficacy was severely restricted in TWIST1‐positive cells. We demonstrated that TWIST1 downregulation restored romidepsin efficacy even in spheroid form, and concomitant SN38 treatment along with romidepsin reproduced the reaction. Conclusions: The current study demonstrated the benefits and concerns of using HDACi for SS treatment in 2D and 3D culture conditions and provided molecular evidence that concomitant treatment with SN38 can overcome drug resistance to HDACi by suppressing TWIST1 expression. [ABSTRACT FROM AUTHOR]

Cellular or tumor dormancy, identified recently as one of the main reasons behind post‐therapy recurrence, can be caused by diverse reasons. Chemotherapy has recently been recognized as one of such reasons. However, in‐depth studies of chemotherapy‐induced dormancy are lacking due to the absence of an in vitro human‐relevant model tailor‐made for such a scenario. This report utilized multicellular breast cancer spheroid to create a primary platform for establishing a chemotherapy‐induced dormancy model. It is observed that extreme chemotherapeutic stress affects invasive and non‐invasive spheroids differently. Non‐invasive spheroids exhibit more resilience and maintain viability and migrational ability, while invasive spheroids display heightened susceptibility and improved tumorigenic capacity. Heterogenous spheroids exhibit increased tumorigenic capacity while show minimal survival ability. Further probing of chemotherapeutically dormant spheroids is needed to understand the molecular mechanism and identify dormancy‐related markers to achieve therapeutic success in the future. [ABSTRACT FROM AUTHOR]

A mannose‐binding protein from the mushroom Agaricus bisporus (Abmb) inhibits the growth of MDA‐MB‐231 cells, which is of an aggressive breast cancer subtype. This ability was observed in a monolayer cell (2D) culture setup, which often is unable to capture changes in cell morphology, polarity and division. That shortcoming may overestimate Abmb potency for its development as a pharmaceutical agent and its use in a therapy. Hence, Abmb's inhibition to the cell growth was performed in the 3D cell (spheroid) culture, which is more representative to the situation in vivo. The result showed that, although the presence of Abmb at ~14.7 μM already disrupted the MDA‐MB‐231 cell morphology in the 2D culture, its presence at ~16.5 μM only ceased the growth of the MDA‐MB‐231 spheroid. Further, Abmb is unique because structurally it belongs to the R‐type lectin (RTL) family; most of mannose‐binding protein is of the C‐type lectin (CTL). As the natural ligand of Abmb is unknown thus the mechanism of action is unclear, Abmb effect on the cancer cells was assessed via observation of the altered expression of genes involved in the Wnt/β‐catenin signalling, which is one of the canonical pathways in the proliferation of cancer cells. The results suggested that Abmb did not alter the pathway upon exerting its anti‐proliferative activity to the MDA‐MB‐231 cells. [ABSTRACT FROM AUTHOR]

We aimed to develop a biocompatible treatment to overcome the limitations of polymethyl methacrylate (PMMA) vertebroplasty for osteoporotic compression fracture patients. We synthesized an injectable hydrogel containing PMMA. Mesenchymal stem cell (MSC) spheroids were included in the injectable PMMA‐doped gel (= PMMA‐doped spheroid gel). In vitro, the osteogenic/anti‐inflammatory effects of the embedded spheroids were investigated by the quantitative real‐time polymerase chain reaction method. In vivo, we used ovariectomy (OVX)‐induced osteoporotic rats with injured femurs to investigate the pain‐relief effects. The OVX rats were divided into four groups according to the materials injected (non, PMMA, PMMA gel, and PMMA‐spheroid gel) into the lesion. The immunofluorescence (IF) intensity levels of painful markers in dorsal root ganglia (DRG) were measured. In vitro, a volumetric ratio of the gel of 8 (gel):2 (PMMA) was non‐cytotoxic for MSCs and promoted the expression of osteogenic/anti‐inflammatory markers. In vivo, the values of several bone parameters in the PMMA‐doped spheroid gel group showed remarkable increases compared to those in the PMMA group. In addition, the IF intensity levels of the painful markers were noticeably decreased in the PMMA‐spheroid gel group. We, therefore, suggest that this treatment can be useful for osteoporotic vertebral compression fracture patients. [ABSTRACT FROM AUTHOR]

The elucidation of the metabolic fate of prohibited substances is crucial for the abuse detection. The human hepatocyte cell line HepG2 can be used to study biotransformation. In order to improve this in vitro model system, we compared the HepG2 spheroid generation using three different techniques: a forced floating, a scaffold‐free and a scaffold‐based method. We characterized the spheroids with regard to the expression levels of the proliferation marker Mki67, the liver‐specific marker albumin and biotransformation enzymes. Moreover, the metandienone metabolite pattern was comparatively analysed by high‐performance liquid chromatography mass spectrometry. With all three techniques, HepG2 spheroids were generated showing a degree of differentiation. The forced floating method resulted in very large spheroids (1 mm in diameter) showing signs of necrosis in the centre and a very low metandienone conversion rate. The spheroids formed by the two other techniques were comparable in size with 0.5 mm in diameter on average. Among the three different 3D cultivation methods, the HepG2 spheroids formed on Matrigel® as extracellular matrix were the most promising regarding biotransformation studies on anabolic androgenic steroids. Prospectively, HepG2 spheroids are a promising in vitro model system to study multidrug setups, drug–drug interactions and the biotransformation of other substance classes. [ABSTRACT FROM AUTHOR]

Background: Tissue fusion is a mechanism involved in the development of the heart, iris, genital tubercle, neural tube, and palate during embryogenesis. Failed fusion of the palatal shelves could result in cleft palate (CP), a common birth defect. Organotypic models constructed of human cells offer an opportunity to investigate developmental processes in the human. Previously, our laboratory developed an organoid model of the human palate that contains human mesenchyme and epithelial progenitor cells to study the effects of chemicals on fusion. Methods: Here, we developed an organoid model more representative of the embryonic palate that includes three cell types: mesenchyme, endothelial, and epithelial cells. We measured fusion by a decrease in epithelial cells at the contact point between the organoids and compared the effects of CP teratogens on fusion and toxicity in the previous and current organoid models. We further tested additional suspect teratogens in our new model. Results: We found that the three-cell-type model is more sensitive to fusion inhibition by valproic acid and inhibitors of FGF, BMP, and TGFβRI/II. In this new model, we tested other suspect CP teratogens and found that nocodazole, topiramate, and Y27632 inhibit fusion at concentrations that do not induce toxicity. Conclusion: This sensitive human three-cell-type organotypic model accurately evaluates chemicals for cleft palate teratogenicity. [ABSTRACT FROM AUTHOR]

Problem: In the cell column of anchoring villi, the cytotrophoblast differentiates into extravillous trophoblast (EVT) and invades the endometrium in contact with maternal immune cells. Recently, chemokines were proposed to regulate the decidual immune response. To investigate the roles of chemokines around the anchoring villi, we examined the expression profiles of chemokines in the first‐trimester trophoblast‐derived Swan71 cells using a three‐dimensional culture model. Method of Study: The gene expressions in the spheroid‐formed Swan71 cells were examined by microarray and qPCR analyses. The protein expressions were examined by immunochemical staining. The chemoattractant effects of spheroid‐formed Swan71 cells were examined by migration assay using monocyte‐derived THP‐1 cells. Results: The expressions of an EVT marker, laeverin, and matrix metalloproteases, MMP2 and MMP9, were increased in the spheroid‐cultured Swan71 cells. Microarray and qPCR analysis revealed that mRNA expressions of various chemokines, CCL2, CCL7, CCL20, CXCL1, CXCL2, CXCL5, CXCL6, CXCL8, and CXCL10, in the spheroid‐cultured Swan71 cells were up‐regulated as compared with those in the monolayer‐cultured Swan71 cells. These expressions were significantly suppressed by hypoxia. Migration assay showed that culture media derived from the spheroid‐formed Swan71 cells promoted THP‐1 cell migration. Conclusion: This study indicated that chemokine expressions in Swan71 cells increase under a spheroid‐forming culture and the culture media have chemoattractant effects. Since three‐dimensional cell assembling in the spheroid resembles the structure of the cell column, this study also suggests that chemokines play important roles in the interaction between EVT and immune cells in their early differentiation stage. [ABSTRACT FROM AUTHOR]

Among several types of brain cancers, glioblastoma multiforme (GBM) is a terminal and aggressive disease with a median survival of 15 months despite the most intensive surgery and chemotherapy. Preclinical models that accurately reproduce the tumor microenvironment are vital for developing new therapeutic alternatives. Understanding the complicated interactions between cells and their surroundings is essential to comprehend the tumor's microenvironment, however the monolayer cell culture approach falls short. Numerous approaches are used to develop GBM cells into tumor spheroids, while scaffold‐based spheroids provides the opportunity to investigate the synergies between cells as well as cells and the matrix. This review summarizes the development of various scaffold‐based GBM spheroid models and the prospective for their use as drug testing systems. [ABSTRACT FROM AUTHOR]

Experimental techniques for patient‐derived cancer stem‐cell organoids/spheroids can be powerful diagnostic tools for personalized chemotherapy. However, establishing their cultures from gastric cancer remains challenging due to low culture efficiency and cumbersome methods. To propagate gastric cancer cells as highly proliferative stem‐cell spheroids in vitro, we initially used a similar method to that for colorectal cancer stem cells, which, unfortunately, resulted in a low success rate (25%, 18 of 71 cases). We scrutinized the protocol and found that the unsuccessful cases were largely caused by the paucity of cancer stem cells in the sampled tissues as well as insufficient culture media. To overcome these obstacles, we extensively revised our sample collection protocol and culture conditions. We then investigated the following second cohort and, consequently, achieved a significantly higher success rate (88%, 29 of 33 cases). One of the key improvements included new sampling procedures for tumor tissues from wider and deeper areas of gastric cancer specimens, which allowed securing cancer stem cells more reproducibly. Additionally, we embedded tumor epithelial pieces separately in both Matrigel and collagen type‐I as their preference to the extracellular matrix was different depending on the tumors. We also added a low concentration of Wnt ligands to the culture, which helped the growth of occasional Wnt‐responsive gastric cancer stem‐cell spheroids without allowing proliferation of the normal gastric epithelial stem cells. This newly improved spheroid culture method may facilitate further studies, including personalized drug‐sensitivity tests prior to drug therapy. [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]

Endochondral ossification is the process of bone formation derived from growing cartilage duringskeletal development. In previous studies, we provoked the osteocyte differentiation of osteoblast precursor cells under a three‐dimensional (3D) culture model. To recapitulate the endochondral ossification, the present study utilized the self‐organized scaffold‐free spheroid model reconstructed by pre‐chondrocyte cells. Within 2‐day cultivation in the absence of the chemically induced chondrogenesis supplements, the chondrocyte marker was greatly expressed in the inner region of the spheroid, whereas the hypertrophic chondrocyte marker was strongly detected in the surface region of the spheroid. Notably, we found out that the gene expression levels of osteocyte markers were also greatly upregulated compared to the conventional 2D monolayer. Moreover, after long‐term cultivation for 28 days, it induced morphological changes in the spheroid, such as cellular hypertrophy and death. In this study, in order to recapitulate the initial stage of the endochondral ossification, we highlighted the potentials of the 3D culture method to drive the hypertrophic chondrocyte differentiation of the pre‐chondrocyte cells. [ABSTRACT FROM AUTHOR]

Tertiary lymphoid structures (TLSs) provide specialized niches for immune cells, resulting in improved prognoses for patients undergoing cancer immunotherapy. Shaping TLS‐like niches may improve anti‐cancer immunity and overcome the current limitations of immune cell‐based immunotherapy. Here, it is shown that stromal vascular fraction (SVF) from adipose tissues can enhance dendritic cell (DC)‐mediated T cell immunity by inducing ectopic T lymphocyte clusters. SVF cells expanded ex vivo have phenotypes and functions similar to those of fibroblastic reticular cells in a secondary lymphoid organ, and their properties can be modulated using three‐dimensional spheroid culture and coculture with DCs spiked with antigen‐loaded iron oxide–zinc oxide core‐shell nanoparticles. Thereby, the combination of SVF spheroids and mature DCs significantly augments T cell recruitment and retention at the injection site. This strategy elicits enhanced antigen‐specific immune response and anti‐tumoral immunity in mice, illustrating the potential for a novel immunotherapeutic design using SVF as a structural scaffold for TLS. [ABSTRACT FROM AUTHOR]

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

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

Colorectal cancer (CRC) is a leading cause of cancer‐related death worldwide. The spheroid colony formation assay is a useful method to identify cancer stem cells (CSCs). Using the DLD‐1 and WiDr CRC cell lines, we performed microarray analyses of spheroid body‐forming and parental cells and demonstrated that aldolase, fructose‐bisphosphate C (ALDOC) was overexpressed in the spheroid body‐forming cells of both lines. Cells transfected with small interfering RNA against ALDOC demonstrated lower proliferation, migration, and invasion compared with negative control cells. Both the number and size of spheres produced by the CRC cells were significantly reduced by ALDOC knockdown. Additionally, inhibition of ALDOC reduced lactate production. Immunohistochemistry was used to analyze ALDOC protein expression in tissues from 135 CRC patients and revealed that 66 (49%) cases were positive for ALDOC. The ALDOC‐positive cases were associated with higher T and M grades and, as determined by Kaplan–Meier analysis, a poorer prognosis. Univariate and multivariate analyses indicated that ALDOC expression was an independent prognostic factor for CRC patients. Furthermore, ALDOC expression was associated with CD44 expression. These results suggest that ALDOC contributes to CRC progression and plays an important role in CSCs derived from CRC. [ABSTRACT FROM AUTHOR]

We employed aqueous solutions of highly‐hydrolyzed (>99+%) poly(vinyl alcohol), PVA, to coat plastic dishes as a method to efficiently induce three‐dimensional (3D) culturing of cells. The coatings were prepared by simple evaporation of 3 wt/vol% solutions of PVA in water and require no additional processing steps after air drying under sterile conditions. The coating allows spheroids to form in solution. Spheroid formation is usually preferable to two‐dimensional (2D) culturing as it creates a more realistic ex vivo model of some human tissues and tumors. Using PVA‐coated cell culture plates, we demonstrated that we can grow reproducibly sized spheroids using several human glioma cell lines, including LN229, U87 MG, and Gli36, and the embryonic kidney cell line, 293T. Spheroids formed on PVA‐coated plates grow as well as on other commercially‐available, low‐attachment plates, and have excellent optical imaging properties. As spheroids, LN229 cells express markers of cancer stem cells. Finally, we confirmed that spheroids generated on PVA‐coated plates are sensitive to molecular perturbations, as increased expression of the cell adhesion molecule PTPμ significantly increased the size of spheroids. The PVA hydrogel layer is an effective tool for creating a more realistic ex vivo culture system than traditional 2D culture and can be used to generate cell spheroids for potential application in drug screening and personalized medicine for diseases such as cancer. [ABSTRACT FROM AUTHOR]

Three‐dimensional (3D) cultures of cancer cells in liquid without extracellular matrix (ECM) offer in vitro models for metastasising conditions such as those in vessels and effusion. However, liquid culturing is often hindered by cell adhesiveness, which causes large cell clumps. We previously described a liquid culture material, LA717, which prevents nonclonal cell adhesion and subsequent clumping, thus allowing clonal growth of spheroids in an anchorage‐independent manner. Here, we examined such liquid culture cancer spheroids for the acquisition of apical–basal polarity, sensitivity to an Akt inhibitor (anticancer drug MK‐2206) and interaction with ECM. The spheroids present apical plasma membrane on the surface, which originated from the failure of polarisation at the single‐cell stage and subsequent defects in phosphorylated ezrin accumulation at the cell boundary during the first cleavage, failing internal lumen formation. At the multicellular stage, liquid culture spheroids presented bleb‐like protrusion on the surface, which was enhanced by the activation of the PI3K/Akt pathway and reduced by PI3K/Akt inhibitors. Liquid culture spheroids exhibited slow proliferation speed and low endogenous pAkt levels compared with gel‐cultured spheroids and 2D‐cultured cells, explaining the susceptibility to the Akt‐inhibiting anticancer drug. Subcutaneous xenografting and in vitro analysis demonstrated low viability and adhesive property of liquid culture spheroids to ECM, while migratory and invasive capacities were comparable with gel‐cultured spheroids. These features agree with the low efficacy of circulating tumour spheroids in the settling step of metastasis. This study demonstrates the feature of anchorage‐independent spheroids and validates liquid cultures as a useful method in cancer spheroid research. [ABSTRACT FROM AUTHOR]

Understanding the ways in which pathogens infect host cells is essential to improve and develop new treatment strategies. This study aimed to generate a novel in vitro infection model by establishing a reproducible 3D spheroid cell culture system that may lead to a reduced need for animals in fish disease research. 2D models (commonly cell lines) cannot replicate many key conditions of in vivo infections, but 3D spheroids have the potential to provide bridging technology between in vivo and in vitro systems. 3D spheroids were generated using cells from rainbow trout (Oncorhynchus mykiss) cell lines, RTG‐2 and RTS‐11. The RTG‐2 spheroids were tested for their potential to be infected upon exposure to Saprolegnia parasitica spores. Positive infiltration of mycelia into the spheroids was verified by confocal microscopy. As a closer analogue of in vivo conditions encountered during infection, the straightforward model developed in this study shows promise as an additional tool that can be used to further our understanding of host–pathogen interactions for Saprolegnia and possibly a variety of other fish pathogens. [ABSTRACT FROM AUTHOR]

The current study evaluated the hypothesis that the administration of spheroidal adipose‐derived stromal/stem cells (ASCs) promotes cell survival and arrests the progression of surgically induced osteoarthritis (OA) in a rat model. We also tested the optimal conditions for spheroid production from ASCs using microwell methods. The formation of ASC spheroids was optimized at a well diameter of 600 μm under cell concentrations of 106cell/ml. When ASC spheroids cultured in 3D were compared with ASCs cultured in 2D monolayer, the cell survival and chondrogenic potential were enhanced while the apoptosis was reduced in ASC spheroids compared with ASCs in 2D monolayer culture. In vivo tracking of fluorescently labeled ASCs in the knee joints of rats with surgically induced OA showed longer fluorescent activity at a higher intensity in ASC spheroids than in ASC single‐cell suspension. When OA‐induced rats treated with ASC injection were sacrificed after 8 weeks, the OARSI score was enhanced in both ASC single‐cell suspension and ASC spheroids compared with negative control, spheroid treatment resulting in a better score than single‐cell treatment. However, injected cells were not detectable from the joints. These finding altogether suggests that ASC spheroids have better in vitro and in vivo survival and chondrogenic potential and exert greater regenerative effects for articular cartilage and arrest the progression of surgically induced OA better than ASCs in single‐cell suspension by the paracrine mode of action. The study findings support the notion of developing cell therapeutics to treat OA based on ASC spheroid forms. [ABSTRACT FROM AUTHOR]

Poor oxygen transport is a major obstacle currently for 3D microtissue culture platforms, which at this time cannot be grown large enough to be truly physiologically relevant and replicate adult human organ functions. To overcome internal oxygen transport deficiencies, oxygenating microgels are formed utilizing perfluorocarbon (PFC) modified chitosan and a highly scalable water‐in‐oil miniemulsion method. Microgels that are on the order of a cell diameter (≈10 µm) are formed allowing them to directly associate with cells when included in 3D spheroid culture, while not being internalized. The presence of immobilized PFCs in these microgels allows for enhancement and tuning of oxygen transport when incorporated into cultured microtissues. As such, it is demonstrated that incorporating oxygenating microgels at ratios ranging from 50:1 to 400:1 (# of cells:# of microgels) into dense human fibroblast‐based spheroids facilitated the growth of larger human cell‐based spheroids, especially at the highest incorporation percentages (50:1), which lacked defined hypoxic cores. Quantification of total double‐stranded (ds)‐DNA, a measure of number of live cells, demonstrated similar results to hypoxia quantification, showing more ds‐DNA due incorporation of oxygenating microgels. Finally, oxygen concentrations are measured at different depths within spheroids directly and confirmed higher oxygen partial pressures due to chitosan‐PFC microspheres. [ABSTRACT FROM AUTHOR]

Acquired chemoresistance is a critical issue for advanced bladder cancer patients during long‐term treatment. Recent studies reveal that a fraction of tumor cells with enhanced tumor‐initiating potential, or cancer stem‐like cells (CSCs), may particularly contribute to acquired chemoresistance and recurrence. Thus, CSC characterization will be the first step towards understanding the mechanisms underlying advanced disease. Here we generated long‐term patient‐derived cancer cells (PDCs) from bladder cancer patient specimens in spheroid culture, which is favorable for CSC enrichment. Pathological features of bladder cancer PDCs and PDC‐dependent patient‐derived xenografts (PDXs) were basically similar to those of their corresponding patients' specimens. Notably, CSC marker aldehyde dehydrogenase 1A1 (ALDH1A1), a critical enzyme that synthesizes retinoic acid (RA), was abundantly expressed in PDCs. ALDH1A1 inhibitors and shRNAs repressed both PDC proliferation and spheroid formation, whereas all‐trans RA could rescue ALDH1A1 shRNA‐suppressed spheroid formation. ALDH inhibitor also reduced the in vivo growth of PDC‐derived xenografts. ALDH1A1 knockdown study showed that tubulin beta III (TUBB3) was one of the downregulated genes in PDCs. We identified functional RA response elements in TUBB3 promoter, whose transcriptional activities were substantially activated by RA. Clinical survival database reveals that TUBB3 expression may associate with poor prognosis in bladder cancer patients. Moreover, TUBB3 knockdown was sufficient to suppress PDC proliferation and spheroid formation. Taken together, our results indicate that ALDH1A1 and its putative downstream target TUBB3 are overexpressed in bladder cancer, and those molecules could be applied to alternative diagnostic and therapeutic options for advanced disease. What's new? Cancer stem‐like cells can contribute to acquired chemoresistance. Studying the characteristics of these cells could yield new ways to thwart chemoresistance. Here, the authors generated 3D cell cultures from patient‐derived bladder cancer cells. They found the cells produced an abundance of aldehyde dehydrogenase 1A1 (ALDH1A1), a stem cell marker. By inhibiting ALDH1A1, they repressed proliferation and spheroid formation. Adding retinoic acid at least partly restored proliferation in ALDH1A1 knockdowns. They also showed that knocking down TUBB3, a target of retinoic acid receptor alpha, likewise repressed proliferation. Thus, they suggest, ALDH1A1 and TUBB3 could be promising therapeutic targets. [ABSTRACT FROM AUTHOR]

Hypoxia‐inducible factor 1 (HIF‐1) is a critical heterodimeric transcription factor for tumor malignancy. Recently, ubiquitin carboxyl‐terminal hydrolase L1 (UCHL1) has been reported to function as a deubiquitinating enzyme for the stabilization of its α subunit (HIF‐1α). In the present study, we showed that UCHL1 inhibition can be an effective therapeutic strategy against HIF‐1‐dependent tumor malignancy. In 2D monolayer culture, a UCHL1 inhibitor suppressed HIF activity and decreased the transcription of HIF downstream genes by inhibiting the UCHL1‐mediated accumulation of HIF‐1α. Phenotypically, UCHL1 inhibition remarkably blocked cell migration. In 3D spheroid culture models, ectopic expression of UCHL1 significantly upregulated malignancy‐related factors such as solidity, volume, as well as viable cell number in an HIF‐1α‐dependent manner. Conversely, inhibition of the UCHL1‐HIF‐1 pathway downregulated these malignancy‐related factors and also abolished UCHL1‐mediated cell proliferation and invasiveness. Finally, inhibition of UCHL1 promoted HIF‐1α degradation and lowered the expression of HIF‐1 target genes in the 3D model, as also observed in 2D monolayer culture. Our research indicates that the UCHL1‐HIF‐1 pathway plays a crucial role in tumor malignancy, making it a promising therapeutic target for cancer chemotherapy. [ABSTRACT FROM AUTHOR]

Aim: To describe an in vitro experimental model of cystic structure formation to conduct research on radicular cyst development. Methodology: To form spheroid structures, various numbers (1 × 104, 5 × 104 or 1 × 105) of epithelial cells (HaCaT and Cal27) were seeded in 96‐well plates previously coated with 1.5% low‐melting agarose. After 24 h, the spheroids were collected, embedded in 3D collagen matrix and transferred to 24‐well plates previously coated with polymerized collagen and kept for up to 21 days. Images of spheroids were captured at each time‐point (1, 5, 9, 15 and 21 days), and samples underwent histological and confocal microscopy analyses. Spheroid area, perimeter and cell dispersion were measured. One‐way Anova was used for statistical analysis. Results: Both epithelial cell lines were able to generate regular and circular spheroids after 24 h of incubation regardless of cell density. Spheroid structures in the collagen matrix were uniform in most samples until day 15, when several spots that appeared to be new cultures were seen. Spheroids from HaCaT were significantly more stable than those from Cal27 (P < 0.05). Starting on the third day, the examination of histological sections revealed a cavity with epithelial lining morphology, similar to a pathological radicular cyst. Conclusions: This study describes an experimental model of cystogenesis in vitro that may be used to test theories and investigates the effects of different growth factors during cyst development and maintenance. [ABSTRACT FROM AUTHOR]

Forkhead box (FOX) proteins constitute a family of transcription factors that are evolutionarily conserved in various species ranging from yeast to humans. These proteins have functions during development as well as in adulthood. To date, many reports have described the functions of FOX family genes in cancer cells, but the role of FOXB2 is not well understood. In one of the pancreas ductal adenocarcinoma cell lines, Panc‐1 cells, we showed here that FOXB2 expression is barely detectable and that CpG islands in the 5′ regions of the FOXB2 are highly methylated. These findings led us to hypothesize that FOXB2 acts as a tumor suppressor. To clarify our hypotheses, we investigated the effects of FOXB2 over‐expression in Panc‐1 cells. We obtained FOXB2 stable transfectants, and these clones exhibited reduced spheroid formation ability. Expression of β‐catenin, which is reported to be over‐expressed in various cancer cells, was highly suppressed in FOXB2 stable transfectants. Moreover, side population (SP) cell fractions, which have a high tumorigenesis and metastatic potential, as well as anchorage‐independent growth ability, were reduced. These results suggest that FOXB2 has the ability to inhibit the malignant characteristics of Panc‐1 in vitro. [ABSTRACT FROM AUTHOR]

Objectives: The periodontal ligament (PDL) has important roles in maintaining homeostasis, wound healing, and regeneration of periodontal tissues by supplying stem/progenitor cells. Periodontal ligament stem cells (PDLSCs) have mesenchymal stem cell (MSC)‐like characteristics and can be isolated from periodontal tissues. The aim of this study was to examine the effect of three‐dimensional spheroid culture on the characteristics of PDLSCs. Material and Methods: Periodontal ligament stem cells were isolated and cultured from healthy teeth, and PDLSC spheroids were formed by pellet culture in polypropylene tubes. The proliferation of PDLSCs in spheroids and conventional two‐dimensional (2D) cultures were examined by immunostaining for Ki67. Cell death and cell size were analyzed using flow cytometry. Gene expression changes were investigated by quantitative real time PCR. Results: Periodontal ligament stem cells spontaneously formed spheroid masses in pellet culture. The size of PDLSC spheroids was inversely proportional to the culture period. Fewer Ki67‐positive cells were detected in PDLSC spheroids compared to those in 2D culture. Flow cytometry revealed an increase in dead cells and a decrease in cell size in PDLSC spheroids. The expression levels of genes related to anti‐inflammation (TSG6, COX2, MnSOD) and angiogenesis (VEGF, bFGF, HGF) were drastically increased by spheroid culture compared to 2D culture. TSG6 gene expression was inhibited in PDLSC spheroids in the presence of the apoptosis signal inhibitor, Z‐VAD‐FMK. Additionally, PDLSC spheroid transplantation into rat periodontal defects did not induce the regeneration of periodontal tissues. Conclusions: We found that spheroid culture of PDLSCs affected several characteristics of PDLSCs, including the expression of genes related to anti‐inflammation and angiogenesis; apoptosis signaling may be involved in these changes. Our results revealed the characteristics of PDLSCs in spheroid culture and have provided new information to the field of stem cell research. [ABSTRACT FROM AUTHOR]

Curcumin is a polyphenolic compound derived from Curcumin longa L. There are growing bodies of evidence revealing the antitumor effect of curcumin in different tumors; although the molecular mechanism behind this inhibition in glioblastoma multiform (GBM) still remains unclear. Here we investigated the antitumor activity of nano micelles curcumin compared with erlotinib in U‐373 cells in monolayer cell cultures and spheroids models. Furthermore, we characterized affecting cell cycle perturbation, as well as apoptosis induction in GBM cells. The antiproliferative activity of nano micelles curcumin and erlotinib were assessed in monolayer and spheroid models. The influence of the cell cycle and expression levels of nuclear factor κB (NF‐κB) and Wnt/β‐catenin pathway was checked. Nano micelles curcumin suppressed cell growth in U‐373 cells via modulation of Wnt and NF‐κB pathways. Moreover, cells developed an early G2/M cell cycle arrest followed by sub‐G1 apoptosis and apoptotic bodies formation posttreatment with nano micelles curcumin and erlotinib. In the core signaling pathways of GBM, nano micelles curcumin either significantly influences the NF‐κB pathway by decreasing p‐65 expression or significantly inhibits the Wnt/β‐catenin pathway by declining cyclin D1 expression. In conclusion, we have shown that nano micelles curcumin effectively prevent proliferation, and invasion of GBM cells through perturbation of Wnt/β‐catenin and NF‐κB pathways, suggesting further investigations on the therapeutic application of this novel anticancer drug in in vivo models. [ABSTRACT FROM AUTHOR]

Eradication of cancer stem‐like cells (CSLCs) are becoming increasingly an important target for new cancer therapies. The ability to study their behavior in vitro will provide the opportunity for high‐throughput testing of more effective treatments. In this study, spheroid‐like structures' formation and enrichment of HT29 CSLCs were evaluated on a wool keratin‐based substrate as a bio‐mimic of natural extracellular matrix (ECM) proteins. The results indicated that culturing on keratin substrate increased spheroid formation ability and radio‐/chemoresistance of HT29 cells. Moreover, cell surface expression of CD133 CSLCs' marker and the mRNA level of stemness genes such as Nanog, Oct4, and c‐MYC were increased. These data suggest that keratin can potentially be used for spheroid‐like structure formation and enrichment of HT29 CSLCs. In addition, it seems that the induction of stemness characteristics on keratin substrate is probably because of the activation of α2β1 integrin signaling pathway. © 2018 Wiley Periodicals, Inc. J Biomed Mater Res Part B: Appl Biomater 107B: 1264–1271, 2019. [ABSTRACT FROM AUTHOR]

Colonic epithelial cells are highly polarised with a lumen‐facing apical membrane, termed the brush border, and a basal membrane in contact with the underlying extracellular matrix (ECM). This polarity is often maintained in cancer tissue in the form of neoplastic glands and has prognostic value. We compared the cellular polarity of several ex vivo spheroid colonic cancer cultures with their parental tumours and found that those grown as non‐attached colonies exhibited apical brush border proteins on their outer cellular membranes. Transfer of these cultures to an ECM, such as collagen, re‐established the centralised apical polarity observed in vivo. The multidrug resistance protein ABCB1 also became aberrantly polarised to outer colony membranes in suspension cultures, unlike cultures grown in collagen, where it was polarised to central lumens. This polarity switch was dependent on the presence of serum or selected serum components, including epidermal growth factor (EGF), transforming growth factor‐β1 (TGF‐β1) and insulin‐like growth factor‐1 (IGF‐1). The apical/basal orientation of primary cancer colon cultures cultured in collagen/serum was modulated by α2β1 integrin signalling. The polarisation of ABCB1 in colonies significantly altered drug uptake and sensitivity, as the outward polarisation of ABCB1 in suspension colonies effluxed substrates more effectively than ECM‐grown colonies with ABCB1 polarised to central lumens. Thus, serum‐free suspension colonies were more resistant to a variety of anti‐cancer drugs than ECM‐grown colonies. In conclusion, the local stroma, or absence thereof, can have profound effects on the sensitivity of colorectal cultures to drugs that are ABCB1 substrates. © 2018 The Authors. The Journal of Pathology published by John Wiley & Sons Ltd on behalf of Pathological Society of Great Britain and Ireland. [ABSTRACT FROM AUTHOR]

Background: Cleft palate (CP) is a common birth defect, occurring in an estimated 1 in 1,000 births worldwide. The secondary palate is formed by paired palatal shelves, consisting of a mesenchymal core with an outer layer of epithelial cells that grow toward each other, attach, and fuse. One of the mechanisms that can cause CP is failure of fusion, that is, failure to remove the epithelial seam between the palatal shelves to allow the mesenchyme confluence. Epidermal growth factor (EGF) plays an important role in palate growth and differentiation, while it may impede fusion. Methods: We developed a 3D organotypic model using human mesenchymal and epithelial stem cells to mimic human embryonic palatal shelves, and tested the effects of human EGF (hEGF) on proliferation and fusion. Spheroids were generated from human umbilical‐derived mesenchymal stem cells (hMSCs) directed down an osteogenic lineage. Heterotypic spheroids, or organoids, were constructed by coating hMSC spheroids with extracellular matrix solution followed by a layer of human progenitor epithelial keratinocytes (hPEKs). Organoids were incubated in co‐culture medium with or without hEGF and assessed for cell proliferation and time to fusion. Results: Osteogenic differentiation in hMSC spheroids was highest by Day 13. hEGF delayed fusion of organoids after 12 and 18 hr of contact. hEGF increased proliferation in organoids at 4 ng/ml, and proliferation was detected in hPEKs alone. Conclusion: Our results show that this model of human palatal fusion appropriately mimics the morphology of the developing human palate and responds to hEGF as expected. [ABSTRACT FROM AUTHOR]

Objective and Background: Human periodontal ligament mesenchymal stem cells (hPDLMSCs) are reported to be responsible for homeostasis and regeneration of periodontal tissue. Although hPDLMSCs are commonly cultured in monolayers, monolayer cultures have been reported as inferior to 3‐dimensional cultures such as spheroids, which are spherical clusters of cells formed by self‐assembly. The aim of this study was to examine the osteogenic phenotype of spheroids of hPDLMSCs, compared with monolayer cultures of hPDLMSC, in vitro and in vivo. Material and Methods: Spheroids were formed using microwell chips that were tagged with polyethylene glycol. Mesenchymal stem cell (MSC) markers in hPDLMSC spheroids were examined by flow cytometer. Real‐time polymerase chain reaction analysis was examined to measure the expressions of stemness markers and osteogenesis‐related genes in monolayer and spheroid‐cultured hPDLMSCs. Immunofluorescence analysis was performed to confirm protein expressions of stemness markers in PDLMSC spheroids. Nodule formation assay, alkaline phosphatase (ALP) activity assay and transplantation assay in a mouse calvarial defect model were performed to confirm the osteogenic potential of hPDLMSC spheroids. To elucidate the mechanism of spheroid culture enhanced osteogenesis in hPDLMSCs with osteoinductive medium (OIM), a small interfering RNA (siRNA) assay targeted with secreted frizzled‐related protein 3 (SFRP3) was examined. The levels of SFRP3 expression in monolayer and spheroid‐cultured hPDLMSCs with OIM were measured by real‐time polymerase chain reaction and western blotting analysis. ALP gene expression and ALP activity were examined in SFRP3‐deficient hPDLMSC spheroids. Results: The hPDLMSC spheroids expressed MSC markers, which were similar to hPDLMSCs grown in monolayer cultures. Intriguingly, the protein and mRNA expressions of transcription factors that regulate “stemness” were significantly increased in hPDLMSC spheroids, compared with hPDLMSCs in monolayer cultures. Nodule formation by hPDLMSCs was significantly increased in spheroid cultures grown with OIM, compared with monolayer‐cultured hPDLMSCs. ALP activity and expression of osteogenesis‐related genes were also significantly enhanced in hPDLMSC spheroids, compared with monolayer cultures. Treatment with hPDLMSC spheroids significantly enhanced new bone formation in a murine calvarial defect model, compared with hPDLMSCs in monolayer culture. Finally, to elucidate mechanisms by which spheroid culture enhances ALP activation in hPDLMSCs grown with OIM, an siRNA assay was used to manipulate expression of SFRP3, a Wnt signaling antagonist. Knockdown of SFRP3 suppressed ALP gene expression in hPDLMSCs grown in OIM; further, it suppressed ALP activity in spheroid culture. These data suggest that the enhancement of osteogenic potential in hPDLMSC spheroids is regulated through SFRP3‐mediated ALP activation. Conclusion: Spheroid cultures of hPDLMSCs may be a novel and useful tool in regenerative medicine. [ABSTRACT FROM AUTHOR]

Abstract: Aim: To evaluate the impact of hypoxia and hypoxia mimetic agents (HMA) on the formation and activity of spheroids by dental pulp cells (DPC). Methodology: DPC on agarose‐coated plates were treated with hypoxia and the HMA dimethyloxallyl glycine (DMOG), desferrioxamine (DFO) and L‐mimosine (L‐MIM). Images of spheroids were taken directly after seeding and at 6 h and 24 h. Spheroid sizes were quantified by area measurement with ImageJ software. Viability was assessed with Live‐Dead staining, MTT and resazurin‐based toxicity assay. Production of VEGF, IL‐8 and SDF‐1 was evaluated using immunoassays. Data were analysed using Kruskal–Wallis test and post hoc Mann–Whitney U‐test. Results: DPC formed spheroids in the presence of hypoxia, HMA and combined treatment with hypoxia and HMA. No pronounced difference in spheroid size was found in the groups treated with hypoxia, DMOG, DFO, L‐MIM and the combination of hypoxia and the HMA relative to their normoxic controls (P > 0.05). Spheroids appeared vital in Live‐Dead and MTT staining and the resazurin‐based toxicity assay. Evaluation of protein production with immunoassays revealed significantly enhanced levels of VEGF and IL‐8 (P < 0.05), but there was no significant effect on SDF‐1 production (P > 0.05). Treatment with a combination of hypoxia and HMA did not further boost VEGF and IL‐8 production (P > 0.05). Conclusions: Pre‐conditioning with hypoxia and HMA increased the pro‐angiogenic capacity of spheroids whilst not interfering with their formation. Pre‐clinical studies will reveal whether pre‐conditioning of spheroids with hypoxia and HMA can effectively improve the efficiency of cell transplantation approaches for regenerative endodontics. [ABSTRACT FROM AUTHOR]

Biological industries commonly rely on bioreactor systems for the large‐scale production of cells. Cell aggregation, clumping, and spheroid morphology of certain suspension cells make their large‐scale culture challenging. Growing stem cells as spheroids is indispensable to retain their stemness, but large spheroids (>500 µm diameter) suffer from poor oxygen and nutrient diffusion, ultimately resulting in premature cell death in the centers of the spheroids. Despite this, most large‐scale bioprocesses do not have an efficient method for dissociating cells into single cells, but rely on costly enzymatic dissociation techniques. Therefore, we tested a proof‐of‐concept fluid shear‐based mechanical dissociator that was designed to dissociate stem cell spheroids and aggregates. Our prototype was able to dissociate cells while retaining high viability and low levels of apoptosis. The dissociator also did not impact long‐term cell growth or spheroid formation. Thus, the dissociator introduced here has the potential to replace traditional dissociation methods. © 2017 American Institute of Chemical Engineers Biotechnol. Prog., 34:293–298, 2018 [ABSTRACT FROM AUTHOR]

Motor neuron diseases such as amyotrophic lateral sclerosis (ALS) are now recognized as multi-system disorders also involving various non-motor neuronal cell types. The precise extent and mechanistic basis of non-motor neuron damage in human ALS and ALS animal models remain however unclear. To address this, we here studied progressive motor neuronopathy ( pmn) mice carrying a missense loss-of-function mutation in tubulin binding cofactor E (TBCE). These mice manifest a particularly aggressive form of motor axon dying back and display a microtubule loss, similar to that induced by human ALS-linked TUBA4A mutations. Using whole nerve confocal imaging of pmn × thy1.2-YFP16 fluorescent reporter mice and electron microscopy, we demonstrate axonal discontinuities, bead-like spheroids and ovoids in pmn suralis nerves indicating prominent sensory neuropathy. The axonal alterations qualitatively resemble those in phrenic motor nerves but do not culminate in the loss of myelinated fibers. We further show that the pmn mutation decreases the level of TBCE, impedes microtubule polymerization in dorsal root ganglion (DRG) neurons and causes progressive loss of microtubules in large and small caliber suralis axons. Live imaging of axonal transport using GFP-tagged tetanus toxin C-fragment (GFP-TTC) demonstrates defects in microtubule-based transport in pmn DRG neurons, providing a potential explanation for the axonal alterations in sensory nerves. This study unravels sensory neuropathy as a pathological feature of mouse pmn, and discusses the potential contribution of cytoskeletal defects to sensory neuropathy in human motor neuron disease. [ABSTRACT FROM AUTHOR]

Nardilysin ( NRDC) is a metalloendopeptidase of the M16 family. We previously showed that NRDC activates inflammatory cytokine signaling, including interleukin-6-signal transducer and activator of transcription 3 ( STAT3) signaling. NRDC has been implicated in the promotion of breast, gastric and esophageal cancer, as well as the development of liver fibrosis. In this study, we investigated the role of NRDC in the promotion of hepatocellular carcinoma ( HCC), both clinically and experimentally. We found that NRDC expression was upregulated threefold in HCC tissue compared to the adjacent non-tumor liver tissue, which was confirmed by immunohistochemistry and western blotting. We also found that high serum NRDC was associated with large tumor size (>3 cm, P = 0.016) and poor prognosis after hepatectomy (median survival time 32.0 vs 73.9 months, P = 0.003) in patients with hepatitis C ( n = 120). Diethylnitrosamine-induced hepatocarcinogenesis was suppressed in heterozygous NRDC-deficient mice compared to their wild-type littermates. Gene silencing of NRDC with mi RNA diminished the growth of Huh-7 and Hep3B spheroids in vitro. Notably, phosphorylation of STAT3 was decreased in NRDC-depleted Huh-7 spheroids compared to control spheroids. The effect of a STAT3 inhibitor (S3I-201) on the growth of Huh-7 spheroids was reduced in NRDC-depleted cells relative to controls. Our results show that NRDC is a promising prognostic marker for HCC in patients with hepatitis C, and that NRDC promotes tumor growth through activation of STAT3. [ABSTRACT FROM AUTHOR]

The multicellular spheroid model partly mimics tumor microenvironments in vivo and has been reported in plenty of studies regarding radiosensitivity. However, clear isolation of quiescent and proliferating cells in live conditions has been quite difficult owing to technical limitations; therefore, comprehensive characterization could not be done thus far. In this study, we succeeded in separately isolating different cell types using a fluorescent ubiquitination-based cell cycle indicator (Fucci) and determining their radiosensitivities. Unexpectedly, proliferating cells were more radioresistant than quiescent cells due to the contact effect when spheroids were disaggregated immediately after irradiation. However, the radiosensitivity of quiescent cells was not influenced by mild hypoxia (hypoxia-inducible factor-1α-positive but pimonidazole-negative), but their radioresistance became similar to that of proliferating cells due to potentially lethal damage repair when disaggregated 24 h after irradiation. The Fucci system further allowed long-term observation of cell kinetics inside of the spheroid following irradiation using real-time confocal fluorescence scanning. Repeated cycles of recruitment from the quiescent to the proliferating phase resulted in cell loss from the outside of the spheroid toward the inside, causing gradual shrinkage. Interestingly, the central region of the spheroid entered a dormant stage approximately 40 days after irradiation and survived for more than 2 months. Using the Fucci system, we were able to comprehensively characterize the radiosensitivity of spheroids for the first time, which highlights the importance of cell cycle kinetics after irradiation in determining the radiosensitivity under tumor microenvironments. [ABSTRACT FROM AUTHOR]

ABSTRACT Tissue engineering constructs to treat intervertebral disc degeneration must adapt to the hypoxic and inflammatory degenerative disc microenvironment. The objective of this study was to determine the effects of two key design factors, cell type and cell configuration, on the regenerative potential of nucleus pulposus cell (NPC) and mesenchymal stem cell (MSC) constructs. Anabolic and catabolic activity was quantified in constructs of varying cell type (NPCs, MSCs, and a 50:50 co-culture) and varying configuration (individual cells and micropellets). Anabolic and catabolic outcomes were both dependent on cell type. Gene expression of Agg and Col2A1, glycosaminoglycan (GAG) content, and aggrecan immunohistochemistry (IHC), were significantly higher in NPC-only and co-culture groups than in MSC-only groups, with NPC-only groups exhibiting the highest anabolic gene expression levels. However, NPC-only constructs also responded to inflammation and hypoxia with significant upregulation of catabolic genes (MMP-1, MMP-9, MMP-13, and ADAMTS-5). MSC-only groups were unaffected by degenerative media conditions, and co-culture with MSCs modulated catabolic induction of the NPCs. Culturing cells in a micropellet configuration dramatically reduced catabolic induction in co-culture and NPC-only groups. Co-culture micropellets, which take advantage of both cell type and configuration effects, had the most immunomodulatory response, with a significant decrease in MMP-13 and ADAMTS-5 expression in hypoxic and inflammatory media conditions. Co-culture micropellets were also found to self-organize into bilaminar formations with an MSC core and NPC outer layer. Further understanding of these cell type and configuration effects can improve tissue engineering designs. © 2016 The Authors. Journal of Orthopaedic Research published by Wiley Periodicals, Inc. on behalf of the Orthopaedic Research Society. J Orthop Res 35:61-73, 2017. [ABSTRACT FROM AUTHOR]

A 3D microtissues using T47D and JIMT-1 cells were generated to analyze tissue-like response of breast cancer cells after combined human epidermal growth factor receptor 2 ( HER2)-targeted treatment and radiation. Following lentiviral knockdown of HER2, we compared growth rate alterations using 2D monolayers, 3D microtissues, and mouse xenografts. Additionally, to model combined therapeutic strategies, we treated HER2-depleted T47D cells and 3D microtissues using trastuzumab (anti- HER2 antibody) in combination with irradiation. Comparison of HER2 knockdown with corresponding controls revealed growth impairment due to HER2 knockdown in T47D 2D monolayers, 3D microtissues, and xenografts (after 2, 12, and ≥40 days, respectively). In contrast, HER2 knockdown was less effective in inhibiting growth of trastuzumab-resistant JIMT-1 cells in vitro and in vivo. Combined administration of trastuzumab and radiation treatment was also analyzed using T47D 3D microtissues. Administration of both, radiation (5 Gy) and trastuzumab, significantly enhanced the growth inhibiting effect in 3D microtissues. To improve the predictive power of potential drugs-as single agents or in combination-here, we show that regarding tumor growth analyses, 3D microtissues are highly comparable to outcomes derived from xenografts. Considering increased limitations for animal experiments on the one hand and strong need of novel drugs on the other hand, it is indispensable to include highly reproducible 3D microtissue platform in preclinical analyses to validate more accurately the capacity of future drug-combined radiotherapy. [ABSTRACT FROM AUTHOR]

The implementation of microfluidic devices within life sciences has furthered the possibilities of both academic and industrial applications such as rapid genome sequencing, predictive drug studies, and single cell manipulation. In contrast to the preferred two-dimensional cell-based screening, three-dimensional (3D) systems have more in vivo relevance as well as ability to perform as a predictive tool for the success or failure of a drug screening campaign. 3D cell culture has shown an adaptive response to the recent advancements in microfluidic technologies which has allowed better control over spheroid sizes and subsequent drug screening studies. In this review, we highlight the most significant developments in the field of microfluidic 3D culture over the past half-decade with a special focus on their benefits and challenges down the lane. With the newer technologies emerging, implementation of microfluidic 3D culture systems into the drug discovery pipeline is right around the bend. [ABSTRACT FROM AUTHOR]

Cytochrome P450 (CYP) induction is a key risk factor of clinical drug-drug interactions that has to bemitigated in the early phases of drug discovery. Three-dimensional (3D) cultures of hepatocytes in vitro have recently emerged as a potentially better platform to recapitulate the in vivo liver structure and to maintain long-term hepatic functions as compared with conventional two-dimensional (2D) monolayer cultures. However, the majority of published studies on 3D hepatocyte models use rat hepatocytes and the response to CYP inducers between rodents and humans is distinct. In the present study, we constructed tethered spheroids on RGD/galactose-conjugated membranes as an in vitro 3D model using cryopreserved human hepatocytes. CYP3A4mRNA expression in the tethered spheroidswas induced to a significantly greater extent than those in the collagen sandwich cultures, indicating the transcriptional regulation was more sensitive to the CYP inducers in the 3D model. Induction of CYP1A2, CYP2B6 and CYP3A4 activities in the tethered spheroids were comparable to, if not higher than that observed in the collagen sandwich cultures. The membrane-based model is readily integrated into multi-well plates for higher-throughput drug testing applications, which might be an alternative model to screen the CYP induction potential in vitro with more physiological relevance. [ABSTRACT FROM AUTHOR]

Cerebellar ataxia commonly occurs in multiple sclerosis, particularly in chronic progressive disease. Previous reports have highlighted both white matter and grey matter pathological changes within the cerebellum; and demyelination and inflammatory cell infiltrates appear commonly. As Purkinje cell axons are the sole output of the cerebellar cortex, understanding pathologic processes within these cells is crucial to develop strategies to prevent their loss and thus reduce ataxia. We studied pathologic changes occurring within Purkinje cells of the cerebellum. Using immunohistochemic techniques, we found changes in neurofilament phosphorylation states within Purkinje cells, including loss of dephosphorylated neurofilament and increased phosphorylated and hyperphosphorylated neurofilament. We also found Purkinje axonal spheroids and Purkinje cell loss, both of which occurred predominantly within areas of leucocortical demyelination within the cerebellar cortex. These changes have important implications for the study of cerebellar involvement in multiple sclerosis and may help design therapies to reduce the burden of ataxia in the condition. [ABSTRACT FROM AUTHOR]

Cancer hypoxia correlates with therapeutic resistance and metastasis, suggesting that hypoxic adaptation is a critical survival advantage for cancer stem cells (CSCs). Hypoxic metabolism, however, may be a disadvantage in aerobic circulation as the extremely low incidence of metastasis-compared to the high circulating tumor-cell numbers (CTCs)-appears to suggest. As rare metastatic CSCs still survive, we searched for a mechanism that protects them from oxygen in circulation. CSCs form multicellular spheroids in vitro from virtually all cancers tested. We asked, therefore, whether cancers also form spheroids in vivo and whether circulating spheroids play a role in metastasis. We used metabolic, apoptotic and hypoxia assays, we measured aerobic barriers and calculated hypoxia vs. spheroid-size correlations. We detected metabolic/oxidative stress in spheroids, we found correlation between stem cell presence and hypoxia and we showed that the size of hypoxic spheroids is compatible with circulation. To detect spheroids in patients, we worked out a new light-scatter flow cytometry blood test and assayed 67 metastatic and control cases. We found in vivo spheroids with positive stem cell markers in cancer blood and they showed exclusive correlation with metastasis. In conclusion, our data suggest that metastatic success depends on CSC-association with in vivo spheroids. We propose that the mechanism involves a portable 'micro-niche' in spheroids that may support CSC-survival/adaptation in circulation. The new assay may establish a potential early marker of metastatic progression. [ABSTRACT FROM AUTHOR]

The spherical geopotential approximation (SGA) used in most meteorological global models assumes a spherical shape for the Earth and its geopotential field, together with a horizontally uniform gravity field (for physical consistency). This approximation has largely been used from the outset and is still used in operational meteorological forecast systems. However, the magnitude of the errors linked to this approximation is debated, especially for long integrations. Consistent mathematical models of the atmosphere in the ellipsoidal geopotential approximation (EGA) have recently been developed, thus allowing a clean assessment of the errors linked to using the SGA. Two types of error may be anticipated: those arising due to geometrical distortions and those arising due to the spatial differences in the magnitude of the gravity field. The shallow-water system, which is the minimal framework in which both types of errors are likely to manifest themselves, is used in this study. The link between the 'pure forecast errors' examined here and the 'numerical weather prediction (NWP) errors' that would arise in a concrete NWP application (with a data assimilation system) is discussed, in relation to the experimental protocol chosen here. Medium-range forecast errors for idealized fully deterministic flows are first considered in a phenomenological way (phase errors, distortions, etc.) and then some widely documented real cases are examined. [ABSTRACT FROM AUTHOR]

Background information During embryonic development, cell death transforms the solid embryonic cell mass into a hollow structure (cavitation), which allows the surviving cells to differentiate into varied tissues and organs around the cavity. This process can be partly reproduced with embryonic stem cells. However, it is unknown if adult stem cell masses have the same ability to cavitate and then differentiate into organs. In this study, we assessed the capacity of human dental pulp stem cells (DPSCs)-derived spheroids to mimic the above-mentioned cavitation and spontaneous differentiation in vitro. Results DPSCs were able to form large-sized spheroids on matrigel in osteogenic medium. Inside the spheroids, cells in the centre showed positive stain to stem cell markers, alkaline phosphatase and STRO-1. Hypoxia and massive cell death were observed in the core of the spheroids. Cavities were formed when the spheroids were cultivated in the osteogenic medium for about 14 days. After 28 days of cultivation, the surviving cells around the cavity spontaneously differentiated into neuronal (28.8%), vascular (33.3%), osteogenic (46.7%) and cartilaginous (72.0%) tissues under the osteogenic medium only. In contrast, when DPSCs-formed cell sheets were folded into giant-sized lumps and cultivated under the same conditions, the folded cell sheets became an entire lumenal structure and failed to differentiate into neuronal, osteogenic and cartilaginous cells. Marker analysis showed that cavitation-related molecules BMP7 and FGF3 expressed on the wall of the cavity in the spheroids, suggesting that the cavitation was functional, whereas cavitation-related molecules were absent in the folded cell sheets. Conclusions DPSC-derived spheroids can mimic the developmental process of cell survival, cavitation and spontaneous multi-differentiation on matrigel under certain conditions. This work allows for functional studies to investigate organ regeneration with human DPSCs in vitro. [ABSTRACT FROM AUTHOR]

We have developed a simple procedure for deriving pure cultures of growing cancer cells from colorectal cancers, including material refrigerated overnight, for pathological characterization and cytotoxicity assays. Forty-six cancers were processed and cultures set up under varying culture conditions. Use of a Rho kinase ( ROCK1) inhibitor markedly increased culture survival, resulting in 80% of samples growing in culture for at least 1 month and beyond. Overnight refrigeration of samples before culture initiation had little effect on success rates, paving the way for cultures to be established for samples collected over wide geographical areas, such as those for clinical trials. Primary cultures demonstrated good correlation for differentiation markers compared to parent cancers, and were highly dynamic in 3D culture. In Matrigel, many colonies formed central lumens, indicating the presence of stem-like cells. Viable colonies in these cultures recapitulated the in vivo generation of carcinoembryonic antigen ( CEA)-positive necrotic/apoptotic debris, much of which was derived from abnormal vacuolated dynamic 'bubble cells' that have not previously been described. Although bubble cells morphologically resembled signet ring cells, a rare cancer subtype, immunostaining suggested that they were most likely derived from terminally differentiated enterocytes. Micro-assays showed that drug toxicity could be measured in these cultures within hours and with sensitivity down to a few hundred cells. Primary cultures derived by our method provide valid in vitro avatars for studying the pathology of cancers in vitro and are amenable to pre-clinical drug testing, paving the way for personalized cancer treatment. Copyright © 2014 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd. [ABSTRACT FROM AUTHOR]

Synovial sarcoma ( SS) is a malignant soft-tissue tumor characterized by the recurrent chromosomal translocation SS18- SSX. Vascular endothelial growth factor ( VEGF)-targeting anti-angiogenic therapy has been approved for soft-tissue sarcoma, including SS; however, the mechanism underlying the VEGF signal for sarcomagenesis in SS is unclear. Here, we show that SS18- SSX directs the VEGF signal outcome to cellular growth from differentiation. Synovial sarcoma cells secrete large amounts of VEGF under spheroid culture conditions in autocrine fashion. SS18- SSX knockdown altered the VEGF signaling outcome, from proliferation to tubular differentiation, without affecting VEGF secretion, suggesting that VEGF signaling promoted cell growth in the presence of SS18- SSX. Thus, VEGF inhibitors blocked both host angiogenesis and spheroid growth. Simultaneous treatment with VEGF and chemokine (C-X-C motif) ( CXC) ligand 12 and CXC receptor 4 inhibitors and/or ifosfamide effectively suppressed tumor growth both in vitro and in vivo. SS18- SSX directs the VEGF signal outcome from endothelial differentiation to spheroid growth, and VEGF and CXC receptor 4 are critical therapeutic targets for SS. [ABSTRACT FROM AUTHOR]

Abstract: The spherical geopotential approximation used in most meteorological global models assumes a spherical shape for the Earth and its geopotential field. Consequently, the deviation of the geoid surface from a sphere, and the observed meridional variations of the apparent gravity are not represented. These two errors are small but their effect on medium or long‐term forecasts is debated because they are systematic and might have cumulative effects. Various formulations with spheroidal iso‐geopotential surfaces have been proposed recently, but none of them combines the advantages of an accurate description for the geopotential field and of horizontal/vertical orthogonal coordinate surfaces. This article proposes a spheroidal coordinate system which meets these two requirements. The transformation and metric factors are defined analytically. The coordinate system is defined as an approximation of orthogonal horizontal/vertical coordinates in which the vertical lines are not exactly orthogonal to true horizontal surfaces. The consequences of this deviation from orthogonality are quantified and upper bounds for the resulting errors are obtained mathematically, i.e. independently of any arbitrary numerical process. The precision of the coordinate can be made as large as desired by raising the truncation of the Taylor series used to approach its exact value, and it is shown that, in practice, truncation values in the range 5 to 8 are appropriate for global numerical weather prediction. [ABSTRACT FROM AUTHOR]

Background Entomopoxviruses ( EVs) form two types of inclusion body: spheroids, which contain virions, and spindles, which do not. The authors tested whether the spindles from a coleopteran EV, Anomala cuprea EV ( ACEV), enhanced the insecticidal activity of a commercial Bacillus thuringiensis ( Bt) formulation and the susceptibility of scarabaeid pest species in Japan to the virus's spheroids, to assess whether ACEV inclusion bodies are potential biological control agents for pest insects. Results Peroral inoculation with both ACEV spindles and the Bt toxin only or the complete Bt formulation shortened the survival and increased the mortality of treated insects compared with those of insects inoculated with Bt without the spindles (8-38 h of decrease in LT50 values among assays). ACEV showed high infectivity to a major scarabaeid pest species in Japanese sugar cane fields. Conclusion The results suggest that spindles or the constituent protein fusolin can be used as a coagent with Bt formulations, and that fusolin coexpression with a Bt toxin in crops might improve the insecticidal efficacy. In addition, the spheroids are potential biocontrol agents for some scarabaeid pests that are not easy to control because of their underground habitation. © 2013 Society of Chemical Industry [ABSTRACT FROM AUTHOR]

Cancer stem cells (CSCs) have been studied intensively in recent years due to their potential importance for understanding neoplastic disease and the design of antitumor therapies. A number of properties attributed to CSCs have been used to define the CSC population, the most important of which is the ability to initiate reproducibly the growth of tumors in vivo. Other assays such as spheroid formation, expression of particular markers and label retention are also used for defining CSCs, although the degree to which these assays invariably reflect the ability to form tumors in vivo remains to be carefully evaluated. Given the importance of correctly defining and isolating CSCs if valid conclusions about their characteristics are to be made, we used syngeneic animal models to compare these different assays. In standard spheroid assays, cell aggregation rather than spheroid growth from single cell suspensions ensued, but aggregation was circumvented by the inclusion of methylcellulose in the medium. Label-retaining subpopulations did not reliably exhibit an enhanced ability to form spheroids and were enriched for senescent cells. Spheroid-forming ability was found to correspond to expression of established CSC markers, although not invariably. Furthermore, spheroid-forming ability was not always reflected in tumor-initiating properties in vivo. Long-term culture of primary mammary tumor cells as adherent monolayers increased their tumor-initiating ability in vivo. This increase was attenuated when the cells were subsequently cultivated as spheroids. Together these data indicate that assays that are widely used to define CSC subpopulations do not invariably reflect tumor-initiating properties in vivo. [ABSTRACT FROM AUTHOR]

The article presents a study which aims to assess vestibular schwannoma (VS) spheroid models for maximum diameter conversion in the cistern. The study used various methods such as magnetic resonance imaging (MRI) reviews, linear measurements and computerized tracing approach. It concludes that the variability in reporting of VS tumor size contributed in the difficulty in reconciling outcomes studies.