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The increasing global cancer burden necessitates the development of new treatment options. Herbal medicine offers a viable alternative to conventional cancer treatments. Numerous studies have shown that 3-dimensional (3D) cell culture more accurately represents tumor characteristics in vivo. Therefore, this study utilized tumor spheroids to explore the therapeutic efficacy of emodin, a natural product-derived bioactive agent. We investigated differences in chemotherapeutic response between A549 cells cultured in 2D versus spheroids, assessing key factors influencing cancer progression, including apoptosis, cell proliferation, cell cycle, migration and invasion. The findings revealed that spheroid cells displayed increased resistance to emodin compared to cells cultured in 2D. Emodin exhibited a more pronounced cytostatic effect in 2D cells, while its cytotoxic effect was more prominent in spheroid cells. Moreover, emodin treatment diminished the migratory and invasive capabilities of the cells. Mechanistic investigations indicated that emodin triggered apoptosis in A549 cells via the mitochondrial apoptotic pathway. Emodin-treated cells exhibited a significant reduction in the phosphorylation of key cancer progression pathways, including JAK2, STAT3, FAK, and ERK, compared to untreated controls. Molecular docking analysis confirmed the interactions of emodin with JAK2 and FAK. These findings suggest that the JAK2/STAT3 and FAK/ERK signaling pathways may serve as critical drivers of the therapeutic effectiveness of emodin in A549 cells., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier Inc. All rights reserved.)

Gastric cancer (GC) is the third leading cause of cancer-related deaths in Japan, underscoring the urgent need for deeper insights into its pathogenesis. Spheroids provide a more realistic and versatile model for studying cancers and cancer stem cells (CSCs). While fructose-bisphosphate aldolase C (ALDOC) has been identified in colorectal cancer spheroids, its role in GC has remained largely unexplored. This study aimed to elucidate the role of ALDOC in GC by performing single-cell and functional analyses of GC spheroids and cell lines, along with immunohistochemistry of 127 GC samples to assess its correlation with CSC markers. Our single-cell analysis revealed upregulation of ALDOC in spheroids, with pseudotime analysis indicating that ALDOC-expressing cells were predominantly undifferentiated and co-expressed LGR5 and CD44. Further investigation into cell-cell interactions suggested that the stem cell state may be maintained by WNT, BMP, and EGF signaling. Functional assays demonstrated that ALDOC knockdown led to a marked reduction in the growth, invasiveness, and spheroid colony formation capacity of GC cell lines. Clinically, ALDOC was detected in the cytoplasm of 56.7% (72/127) of GC cases, and high ALDOC expression was significantly associated with poor overall survival ( p < 0.01), and was an independent prognostic factor. Moreover, a significant association between ALDOC and CD44 expression in GC ( p = 0.031). Conclusively, our findings identify ALDOC as a crucial prognostic marker and provide new insights into GC pathogenesis., Competing Interests: VThe authors declare that there are no conflicts of interest., (2024 The Japan Society of Histochemistry and Cytochemistry.)

Intravascular large B-cell lymphoma (IVLBCL) is a rare type of extranodal large B-cell lymphoma that is characterized by the proliferation of lymphoma cells in the lumina of small vessels. Recent progress uncovering the genetic characteristics associated with MYD88/CD79B mutations has stimulated interest in the use of drugs targeting B-cell receptor signaling, including Bruton's tyrosine kinase. However, difficulties in culturing ex vivo IVLBCL cells has hampered research on the development of novel therapies. In the present study, we demonstrated the establishment of an ex vivo culture system of IVLBCL cells obtained from patient-derived xenograft (PDX) models. The spheroid culture enabled us to culture IVLBCL PDX cells for more than 10 days and to explore the efficacy of drug treatments acting on these cells. We found that carfilzomib and ibrutinib were effective for treating IVLBCL in ex vivo experiments and conducted in vivo analyses to assess the efficacy of these drugs. Although the efficacy of carfilzomib was difficult to confirm due to its toxicity in our models, ibrutinib showed comparable efficacy to a standard combination of chemotherapy drugs. Together, our data provide a new culture method for IVLBCL PDX cells and a rationale for translating ibrutinib to clinical use in IVLBCL patients., (© 2024 The Author(s). Cancer Science published by John Wiley & Sons Australia, Ltd on behalf of Japanese Cancer Association.)

Mouse neuronal CAD 5 cell line effectively propagates various strains of prions. Previously, we have shown that it can also be differentiated into the cells morphologically resembling neurons. Here, we demonstrate that CAD 5 cells chronically infected with prions undergo differentiation under the same conditions. To make our model more realistic, we triggered the differentiation in the 3D culture created by gentle rocking of CAD 5 cell suspension. Spheroids formed within 1 week and were fully developed in less than 3 weeks of culture. The mature spheroids had a median size of ~300 μm and could be cultured for up to 12 weeks. Increased expression of differentiation markers GAP 43, tyrosine hydroxylase, β-III-tubulin and SNAP 25 supported the differentiated status of the spheroid cells. The majority of them were found in the G0/G1 phase of the cell cycle, which is typical for differentiated cells. Moreover, half of the PrP C on the cell membrane was N-terminally truncated, similarly as in differentiated CAD 5 adherent cells. Finally, we demonstrated that spheroids could be created from prion-infected CAD 5 cells. The presence of prions was verified by immunohistochemistry, western blot and seed amplification assay. We also confirmed that the spheroids can be infected with the prions de novo. Our 3D culture model of differentiated CAD 5 cells is low cost, easy to produce and cultivable for weeks. We foresee its possible use in the testing of anti-prion compounds and future studies of prion formation dynamics., (© 2024 Federation of European Neuroscience Societies and John Wiley & Sons Ltd.)

In hyperparathyroidism (hyperPTH), excessive amounts of PTH are secreted, interfering with calcium regulation in the body. Several drugs can control the disease's side effects, but none of them is an alternative treatment to surgery. Therefore, new drug candidates are necessary. In this study, three computationally repositioned drugs, DG 041, IMD 0354, and cucurbitacin I, are evaluated in an in vitro model of hyperPTH. First, we integrated publicly available transcriptomics datasets to propose drug candidates. Using 3D spheroids derived from a single primary hyperPTH patient, we assessed their in vitro efficacy. None of the proposed drugs affected the viability of healthy cell control (HEK293) or overactive parathyroid cells at the level of toxicity. This behavior was attributed to the non-cancerous nature of the parathyroid cells, establishing the hyperPTH disease model. Cucurbitacin I and IMD 0354 exhibited a slight inverse relationship between increased drug concentrations and cell viability, whereas DG 041 increased viability. Based on these results, further studies are needed on the mechanism of action of the repurposed drugs, including determining the effects of these drugs on cellular PTH synthesis and secretion and on the metabolic pathways that regulate PTH secretion., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2024 Elsevier B.V. All rights reserved.)

Cell therapy using chondrocytes has shown promise for cartilage regeneration, but maintaining functional characteristics during in vitro culture and ensuring survival after transplantation are challenges. Three-dimensional (3D) cell culture methods, such as spheroid culture, and hydrogels can improve cell survival and functionality. In this study, a new method of culturing spheroids using hyaluronic acid (HA) microparticles was developed. The spheroids mixed with HA microparticles effectively maintained the functional characteristics of chondrocytes during in vitro culture, resulting in improved cell survival and successful cartilage formation in vivo following transplantation. This new method has the potential to improve cell therapy production for cartilage regeneration., Competing Interests: Declaration of competing interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2023. Published by Elsevier Ltd.)

The use of laboratory animals in research has been extensively criticized. While most of the critique has been centered around the ethical aspect, also the economic and scientific aspects have been frequently mentioned as points of concern. As a result, the use of alternative methods has gradually become more enticing. The most used alternatives to laboratory animals are the 2D monolayer cell cultures. However, the limited translatability of these monolayer cell cultures to in vivo has led to the development of 3D cell cultures that are believed to better capture the in vivo physiology and pathology. Here we report on the development of a physiologically more relevant 3D cell model (spheroids) comprised of human bronchial epithelial (16HBE14o-) cells, for use in respiratory toxicity research. Culturing 16HBE14o-cells as hanging-drops led to the formation of stable spheroids which showed an increased expression of CLDN1 when compared to 2D monolayer cultured cells. In addition, cell-cycle analysis revealed an increased sub-G0 population and signs of G0/G1 arrest in spheroids. Afterwards, standard operating procedures (SOPs) were established, and existing protocols optimized, for compatibility with spheroids. Spheroids were successfully used to assess cytotoxicity, genotoxicity, apoptosis/necrosis, and oxidative stress after exposure to known cytotoxic or genotoxic compounds. The development of the bronchial epithelial spheroids and the establishment of SOPs can contribute to a more reliable toxicity assessment of chemicals and may aid in bridging the gap between in vivo and in vitro experiments., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Purpose: This study aimed to compare the radiosensitizing effect of the PARP inhibitor, Olaparib, between proton and X-rays irradiations in BRCA-proficient breast cancer (BC) cells., Methods: Two BRCA-proficient BC cell lines, MDA-MB-231 and T47D BC, were used. Cell proliferation was assessed using the CCK-8 assay, and radiosensitivity was determined through the clonogenic survival assay. Flow cytometry was employed to analyze cell cycle distribution and apoptosis. The kinetics of DNA damage repair were evaluated using γH2AX immunofluorescence imaging and the comet assay. Tumor spheroid assays were conducted to test radiosensitivity in a three-dimensional culture condition., Results: Olaparib sensitized both MDA-MB-231 and T47D cells to proton and X-ray irradiation in the clonogenic assay. MDA-MB-231 cells exhibited a higher dose enhancement factor for Olaparib than T47D cells. Olaparib increased radiation-induced G2/M cell cycle arrest and apoptosis specifically in MDA-MB-231 cells. γH2AX immunostaining and the comet assay showed Olaparib augmented radiation-induced DNA damage and apoptosis. The enhancement effect of Olaparib was more pronounced in proton irradiation than in X-ray irradiation, particularly in MDA-MB-231 cells than T47D cells. Both radiation and Olaparib dose-dependently inhibited spheroid growth in both cell lines. The synergy scores demonstrated that Olaparib interacted more strongly with protons than X-rays. The addition of an ATR inhibitor further enhanced Olaparib-induced proton radiosensitization in MDA-MB-231 cells., Conclusion: This study found that Olaparib enhanced radiation efficacy in BRCA-proficient breast cancer cells, with a more pronounced effect observed with proton irradiation compared to X-ray irradiation. Combining Olaparib with an ATR inhibitor increased the radiosensitizing effect of protons., (© 2023. The Author(s), under exclusive licence to Springer Science+Business Media, LLC, part of Springer Nature.)

Validated in vitro assays for testing non-genotoxic carcinogenic potential of chemicals are currently not available. Consequently, the two-year rodent bioassay remains the gold standard method for the identification of these chemicals. Transcriptomic and proteomic analyses have provided a comprehensive understanding of the non-genotoxic carcinogenic processes, however, functional changes induced by effects at transcriptional and translational levels have not been addressed. The present study was set up to test a number of proposed in vitro biomarkers of non-genotoxic hepatocarcinogenicity at the functional level using a translational 3-dimensional model. Spheroid cultures of human hepatocytes and stellate cells were exposed to 5 genotoxic carcinogenic, 5 non-genotoxic carcinogenic, and 5 non-carcinogenic chemical compounds and assessed for oxidative stress, mitochondrial dysfunction, endoplasmic reticulum stress, apoptosis, and inflammation. The spheroid model could capture many of these events triggered by the genotoxic carcinogenic chemicals, particularly aflatoxin B1 and hydroquinone. Nonetheless, no clear distinction could be made between genotoxic and non-genotoxic hepatocarcinogenicity. Therefore, spheroid cultures of human liver cells may be appropriate in vitro tools for mechanistic investigation of chemical-induced hepatocarcinogenicity, however, these mechanisms and their read-outs do not seem to be eligible biomarkers for detecting non-genotoxic carcinogenic chemicals., (© 2023. The Author(s), under exclusive licence to Springer-Verlag GmbH Germany, part of Springer Nature.)

Problem: Endometriosis is a proliferative disease characterized by cytokine-induced inflammation. The objective of this study was to assess cell growth and PGE 2 production induced by TNF-α in endometriotic stromal cells (ESCs) in spheroid cell culture and to identify the signaling pathway involved with a view to finding new therapeutic targets for endometriosis., Method of Study: Tissue samples were collected from patients with and without endometriosis. ESCs were isolated from ovarian endometrioma (OE). Gene expression was evaluated by real-time PCR and DNA microarray analysis, the proliferative effect on ESCs by WST-8 assay, and PGE 2 production by ELISA. Protein phosphorylation was detected using western blotting., Results: COX-2, aromatase and VEGFA mRNA expression and PGE 2 production were significantly elevated in spheroid cell cultures compared to monolayer cell cultures. TNF-α receptor (TNFR) 1 and TNFR2 mRNA was also significantly increased. TNF-α promoted the proliferation and PGE 2 production of ESCs in spheroid cell cultures significantly more than in monolayer cell cultures. TNF-α increased the expression of several genes related to the pathophysiology of endometriosis in spheroid ESCs. DNA microarray analysis revealed that the Tpl2 gene, which codes for a MAPK upstream of MEK, was upregulated in OE and endometrium with endometriosis compared to normal endometrium. TNF-α increased the phosphorylation and expression of Tpl2 and MEK, and Tpl2 and MEK inhibitors inhibited TNF-α-induced proliferation and PGE 2 production in spheroid ESCs., Conclusion: The Tpl2-MEK signaling pathway may play a critical role in the cell growth and PGE 2 production induced by TNF-α in spheroid ESCs., (© 2023 John Wiley & Sons A/S. Published by John Wiley & Sons Ltd.)

Background: Cancer stem cells (CSCs), subpopulations of cancer cells, are responsible for tumor progression, metastasis, and relapse. Changes in amino acid metabolism are linked to breast cancer recurrence and metastasis., Aims: This study aimed to evaluate the changes in the amino acid profile in MCF-7 and MDA-MB-231 cells during spheroid formation to discover the specific metabolic properties in CSCs., Methods: MCF-7 and MDA-MB-231 breast cancer cells were cultured as spheroids and evaluated to characterize their CSC properties. The characteristics of CSC were evaluated by examining the expression of CSC markers and conducting drug resistance assays. In addition, amino acid profile change during the enrichment of breast cancer stem cells in the spheroids was investigated by high-performance liquid chromatography (HPLC)., Results: The results indicated that out of 20 different amino acids analyzed, 19 of them decreased during the spheroid formation process. Alanine, lysine, phenylalanine, threonine, and glycine showed significant reductions in the conditioned media of both cell lines in the spheroid form compared to the monolayer cells. Only one of the amino acids increased in MCF-7 and MDA-MB-231 spheroids (histidine and serine, respectively)., Conclusion: Our results suggest that certain amino acids identified in this study can be used for a better understanding of the molecular mechanisms associated with breast cancer stem cell formation., (© 2023 The Authors. Cancer Reports published by Wiley Periodicals LLC.)

The need for maintaining cell-spheroid viability and function within high-density cultures is unmet for various clinical and experimental applications, including cell therapies. One immediate application is for transplantation of pancreatic islets, a clinically recognized treatment option to cure type 1 diabetes; islets are isolated from a donor for subsequent culture prior to transplantation. However, high seeding conditions cause unsolicited fusion of multiple spheroids, thereby limiting oxygen diffusion to induce hypoxic cell death. Here we introduce a culture dish incorporating a micropyramid-patterned surface to prevent the unsolicited fusion and oxygen-permeable bottom for optimal oxygen environment. A 400 µ m-thick, oxygen-permeable polydimethylsiloxane sheet topped with micropyramid pattern of 400 µ m-base and 200 µ m-height was fabricated to apply to the 24-well plate format. The micropyramid pattern separated the individual pancreatic islets to prevent the fusion of multiple islets. This platform supported the high oxygen demand of islets at high seeding density at 260 islet equivalents cm -2 , a 2-3-fold higher seeding density compared to the conventional islet culture used in a preparation for the clinical islet transplantations, demonstrating improved islet morphology, metabolism and function in a 4 d-culture. Transplantation of these islets into immunodeficient diabetic mice exhibited significantly improved engraftment to achieve euglycemia compared to islets cultured in the conventional culture wells. Collectively, this simple design modification allows for high-density cultures of three-dimensional cell spheroids to improve the viability and function for an array of investigational and clinical replacement tissues., (© 2022 IOP Publishing Ltd.)

Sjӧgren's syndrome (SS) is a systemic autoimmune disease that targets the exocrine glands, resulting in impaired saliva and tear secretion. To date, type I interferons (I-IFNs) are increasingly recognized as pivotal mediators in SS, but their endogenous drivers have not been elucidated. Here, we investigate the role of mitochondrial double-stranded RNAs (mt-dsRNAs) in regulating I-IFNs and other glandular phenotypes of SS. We find that mt-dsRNAs are elevated in the saliva and tears of SS patients (n = 73 for saliva and n = 16 for tears) and in salivary glands of non-obese diabetic mice with salivary dysfunction. Using the in-house-developed 3D culture of immortalized human salivary gland cells, we show that stimulation by exogenous dsRNAs increase mt-dsRNAs, activate the innate immune system, trigger I-IFNs, and promote glandular phenotypes. These responses are mediated via the Janus kinase 1 (JAK1)/signal transducer and activator of transcription (STAT) pathway. Indeed, a small chemical inhibitor of JAK1 attenuates mtRNA elevation and immune activation. We further show that muscarinic receptor ligand acetylcholine ameliorates autoimmune characteristics by preventing mt-dsRNA-mediated immune activation. Last, direct suppression of mt-dsRNAs reverses the glandular phenotypes of SS. Altogether, our study underscores the significance of mt-dsRNA upregulation in the pathogenesis of SS and suggests mt-dsRNAs as propagators of a pseudo-viral signal in the SS target tissue., Competing Interests: S.G.I. is a co-inventor of the polymeric thin-film-based 3D culture used in this study and is currently filing a patent for it. The authors declare no competing interests., (© 2022 The Author(s).)

Bone health-targeting drug development strategies still largely rely on inferior 2D in vitro screenings. We aimed at developing a scaffold-free progenitor cell-based 3D biomineralization model for more physiological high-throughput screenings. MC3T3-E1 pre-osteoblasts were cultured in α-MEM with 10% FCS, at 37 °C and 5% CO 2 for up to 28 days, in non-adherent V-shaped plates to form uniformly sized 3D spheroids. Osteogenic differentiation was induced by 10 mM β-glycerophosphate and 50 µg/mL ascorbic acid. Mineralization stages were assessed through studying expression of marker genes, alkaline phosphatase activity, and calcium deposition by histochemistry. Mineralization quality was evaluated by Fourier transformed infrared (FTIR) and scanning electron microscopic (SEM) analyses and quantified by micro-CT analyses. Expression profiles of selected early- and late-stage osteoblast differentiation markers indicated a well-developed 3D biomineralization process with strongly upregulated Col1a1 , Bglap and Alpl mRNA levels and type I collagen- and osteocalcin-positive immunohistochemistry (IHC). A dynamic biomineralization process with increasing mineral densities was observed during the second half of the culture period. SEM-Energy-Dispersive X-ray analyses (EDX) and FTIR ultimately confirmed a native bone-like hydroxyapatite mineral deposition ex vivo. We thus established a robust and versatile biomimetic, and high-throughput compatible, cost-efficient spheroid culture model with a native bone-like mineralization for improved pharmacological ex vivo screenings.

Cell dissemination during tumor development is a characteristic of cancer metastasis. Dissemination from three-dimensional spheroid models on extracellular matrices designed to mimic tissue-specific physiological microenvironments may allow us to better elucidate the mechanism behind cancer metastasis and the response to therapeutic agents. The orientation of fibrillar collagen plays a key role in cellular processes and mediates metastasis through contact-guidance. Understanding how cells migrate on aligned collagen fibrils requires in vitro assays with reproducible and standardized orientation of collagen fibrils on the macro-to-nanoscale. Herein, we implement a spheroid-based migration assay, integrated with a fibrillar type I collagen matrix, in a manner compatible with high throughput image acquisition and quantitative analysis. The migration of highly proliferating U2OS osteosarcoma cell spheroids onto an aligned fibrillar type I collagen matrix was quantified. Cell dissemination from the spheroid was polarized with increased invasion in the direction of fibril alignment. The resulting area of cell dissemination had an aspect ratio of 1.2 ± 0.1 and an angle of maximum invasion distance of 5° ± 44° relative to the direction of collagen fibril alignment. The assay described here can be applied to a fully automated imaging and analysis pipeline for the assessment of tumor cell migration with high throughput screening., Competing Interests: Declaration of Competing Interest The authors declare that they have no known competing financial interests or personal relationships that could have appeared to influence the work reported in this paper., (Copyright © 2022. Published by Elsevier Inc.)

Epidermal growth factor receptor (EGFR) upregulation is a typical characteristic of head and neck squamous cell carcinoma (HNSCC). However, tyrosine kinase inhibitors have not yet been able to achieve enough therapeutic benefit in clinical trials to justify their use in standard therapy regimens. At present, little is known about the reasons for this treatment failure. In the present study, the HNSCC cell lines UM-SCC-11B and UM-SCC-22B were tested for their response to tyrosine kinase inhibitors (TKI) under 2D and 3D cell culture conditions. Absorption and luciferase-based viability assays were used for this, as well as optical evaluation via fluorescence microscopy. In addition, EGFR and HER3 expression as well as the downstream signalling pathways PI3K/AKT/mTOR and RAS/RAF/MEK/ERK were investigated using western blotting. Cell line UM-SCC-11B revealed a strong resistance to lapatinib under 3D cell culture conditions, while a good response to TKI therapy was observed under 2D cell culture conditions. An associated overexpression of phosphorylated HER3 under 3D cell culture conditions offered a plausible explanation for the altered treatment response. The results of the present study represent an idea of how signalling mechanisms of cancer cells can be changed using different cell culture methods. Overall, 3D cell culture could be an important component in the analysis of resistance mechanisms in cancer therapy., Competing Interests: The authors declare that they have no competing interests., (Copyright: © Heid et al.)

In vitro 3D cancer spheroids (tumoroids) exhibit a drug resistance profile similar to that found in solid tumors. 3D spheroid culture methods recreate more physiologically relevant microenvironments for cells. Therefore, these models are more appropriate for cancer drug screening. We have recently developed a protocol for MCF-7 cell spheroid culture, and used this method to test the effects of different types of drugs on this estrogen-dependent breast cancer cell spheroid. Our results demonstrated that MCF-7 cells can grow spheroid in medium using a low attachment plate. We managed to grow one spheroid in each well, and the spheroid can grow over a month, the size of the spheroid can grow over a hundred times in volume. Our targeted drug experimental results suggest that estrogen sulfotransferase, steroid sulfatase, and G protein-coupled estrogen receptor may play critical roles in MCF-7 cell spheroid growth, while estrogen receptors α and β may not play an essential role in MCF-7 spheroid growth. Organoids are the miniatures of in vivo tissues and reiterate the in vivo microenvironment of a specific organ, best fit for the in vitro studies of diseases and drug development. Tumoroid, developed from cancer cell lines or patients' tumor tissue, is the best in vitro model of in vivo tumors. 3D spheroid technology will be the best future method for drug development of cancers and other diseases. Our reported method can be developed clinically to develop personalized drugs when the patient's tumor tissues are used to develop a spheroid culture for drug screening., Competing Interests: Conflicts of Interest The authors declare no potential conflicts of interest.

Background: Quercetin (QCT) is a dietary flavonoid with many beneficial effects (e.g., antioxidant, antiaging, antidiabetic, antifungal effects, and regulation of gastrointestinal motor activity in human); furthermore, it induces apoptosis, cell cycle arrest, and differentiation., Objective: The apoptotic effects of OCT were investigated on SW480 human colon cancer cell lines in monolayer and spheroid cultures., Methods: Quercetin (40-200 μM) was applied, and Inhibitory Concentration (IC50) doses were determined for three time intervals (24, 48, and 72 h). The effective dose was determined and applied for analyses, including staining with BrdU to investigate cell proliferation, terminal deoxynucleotidyl transferase dUTP nick and labeling (TUNEL) to investigate apoptosis, and caspase-3 and Apoptosis Inducing Factor (AIF) to investigate caspase-dependent or independent apoptotic pathways., Results: The effective dose of QCT was determined to be 200 μM and was found to induce apoptosis and inhibit cell proliferation at 24, 48, and 72 h,both in 2D and 3D cultures. Significant increases were observed in both caspase-3 and AIF staining, but cells showed greater caspase-3 staining compared with AIF staining at all time intervals (p<0.05)., Conclusion: The QCT treatment groups showed more cell death and less cell growth compared with the untreated control groups in both 2D and 3D cultures of SW480 cell lines. The results suggest that quercetin induces apoptosis, inhibits cell proliferation, and has a protective role against colon cancer. However, further studies are needed to clarify its mechanism of action., (Copyright© Bentham Science Publishers; For any queries, please email at epub@benthamscience.net.)

Incorporation of extracellular matrix (ECM) and hydrogel in microfluidic 3D cell culture platforms is important to create a physiological microenvironment for cell morphogenesis and to establish 3D co-culture models by hydrogel compartmentalization. Here, we describe a simple and scalable ECM patterning method for microfluidic cell cultures by achieving hydrogel confinement due to the geometrical expansion of channel heights (stepped height features) and capillary burst valve (CBV) effects. We first demonstrate a sequential "pillar-free" hydrogel patterning to form adjacent hydrogel lanes in enclosed microfluidic devices, which can be further multiplexed with one to two stepped height features. Next, we developed a novel "spheroid-in-gel" culture device that integrates (1) an on-chip hanging drop spheroid culture and (2) a single "press-on" hydrogel confinement step for rapid ECM patterning in an open-channel microarray format. The initial formation of breast cancer (MCF-7) spheroids was achieved by hanging a drop culture on a patterned polydimethylsiloxane (PDMS) substrate. Single spheroids were then directly encapsulated on-chip in individual hydrogel islands at the same positions, thus, eliminating any manual spheroid handling and transferring steps. As a proof-of-concept to perform a spheroid co-culture, endothelial cell layer (HUVEC) was formed surrounding the spheroid-containing ECM region for drug testing studies. Overall, this developed stepped height-based hydrogel patterning method is simple to use in either enclosed microchannels or open surfaces and can be readily adapted for in-gel cultures of larger 3D cellular spheroids or microtissues.

Background: Anisomycin, a potential anticancer therapeutic drug, exerts an antitumor effect on melanoma cells at a lower concentration than that required for other cancer cells. However, the molecular mechanisms remain unclear., Materials and Methods: The sensitivity to and cytotoxicity of anisomycin, as well as the effects of anisomycin on glucose metabolism and relative mRNA expression of senescence- and cancer-associated genes, were studied using B16 mouse melanoma cells., Results: The viability of anisomycin-treated cells decreased in a concentration-dependent manner, and the growth of cell spheroids was suppressed by 50 nM anisomycin. Glucose metabolism was reduced by anisomycin treatment, and the mRNA expression of genes responsible for growth inhibition, such as p21, p53 and Txnip was upregulated., Conclusion: The results suggest that anisomycin may be a promising future anticancer drug that is effective at low concentrations against melanoma by reducing glucose metabolism, causing cell senescence-like phenomena., (Copyright © 2021 International Institute of Anticancer Research (Dr. George J. Delinasios), All rights reserved.)

Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation and bone regeneration capacity of mesenchymal stromal cells (MSC). Gingiva-derived progenitor cells (GPC) represent a less invasive alternative to bone marrow MSC (BMSC) for clinical applications. The aim of this study was to test the in vivo bone forming potential of human GPC and BMSC cultured as 3D spheroids or dissociated cells (2D). 2D and 3D cells encapsulated in constructs of human platelet lysate hydrogels (HPLG) and 3D-printed poly (L-lactide-co-trimethylene carbonate) scaffolds (HPLG-PLATMC) were implanted subcutaneously in nude mice; cell-free HPLG-PLATMC constructs served as a control. Mineralization was assessed using micro-computed tomography (µCT), histology, scanning electron microscopy (SEM) and in situ hybridization (ISH). After 4-8 weeks, µCT revealed greater mineralization in 3D-BMSC vs. 2D-BMSC and 3D-GPC ( p < 0.05), and a similar trend in 2D-GPC vs. 2D-BMSC ( p > 0.05). After 8 weeks, greater mineralization was observed in cell-free constructs vs. all 2D- and 3D-cell groups ( p < 0.05). Histology and SEM revealed an irregular but similar mineralization pattern in all groups. ISH revealed similar numbers of 2D and 3D BMSC/GPC within and/or surrounding the mineralized areas. In summary, spheroid culture promoted ectopic mineralization in constructs of BMSC, while constructs of dissociated GPC and BMSC performed similarly. The combination of HPLG and PLATMC represents a promising scaffold for bone tissue engineering applications., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shanbhag, Kampleitner, Mohamed-Ahmed, Yassin, Dongre, Costea, Tangl, Stavropoulos, Bolstad, Suliman and Mustafa.)

Background: Three-dimensional (3D) spheroid culture can promote the osteogenic differentiation of bone marrow mesenchymal stromal cells (BMSC). 3D printing offers the possibility to produce customized scaffolds for complex bone defects. The aim of this study was to compare the potential of human BMSC cultured as 2D monolayers or 3D spheroids encapsulated in constructs of 3D-printed poly-L-lactide-co-trimethylene carbonate scaffolds and modified human platelet lysate hydrogels (PLATMC-HPLG) for bone regeneration., Methods: PLATMC-HPLG constructs with 2D or 3D BMSC were assessed for osteogenic differentiation based on gene expression and in vitro mineralization. Subsequently, PLATMC-HPLG constructs with 2D or 3D BMSC were implanted in rat calvarial defects for 12 weeks; cell-free constructs served as controls. Bone regeneration was assessed via in vivo computed tomography (CT), ex vivo micro-CT and histology., Results: Osteogenic gene expression was significantly enhanced in 3D versus 2D BMSC prior to, but not after, encapsulation in PLATMC-HPLG constructs. A trend for greater in vitro mineralization was observed in constructs with 3D versus 2D BMSC (p > 0.05). In vivo CT revealed comparable bone formation after 4, 8 and 12 weeks in all groups. After 12 weeks, micro-CT revealed substantial regeneration in 2D BMSC (62.47 ± 19.46%), 3D BMSC (51.01 ± 24.43%) and cell-free PLATMC-HPLG constructs (43.20 ± 30.09%) (p > 0.05). A similar trend was observed in the histological analysis., Conclusion: Despite a trend for superior in vitro mineralization, constructs with 3D and 2D BMSC performed similarly in vivo. Regardless of monolayer or spheroid cell culture, PLATMC-HPLG constructs represent promising scaffolds for bone tissue engineering applications., (© 2021. The Author(s).)

Suspended spheroid culture using ultralow attachment plates (ULAPs) is reported to effect corneal fibroblast reprogramming. Polydimethylsiloxane (PDMS), with hydrophobic and soft substrate properties, facilitates adherent spheroid formation that promotes cellular physical reprogramming into stem-like cells without using transcription factors. However, it is still unknown whether the biophysical properties of PDMS have the same effect on adult human corneal keratocyte reprogramming. Here, PDMS and essential 8 (E8) medium were utilized to culture keratocyte spheroids and fibroblast spheroids, and the reprogramming results were compared. We provide insights into the probable mechanisms of the PDMS effect on spheroids. qPCR analysis showed that the expression of some stem cell marker genes ( OCT4, NANOG, SOX2, KLF4, CMYC , ABCG2 and PAX6 ) was significantly greater in keratocyte spheroids than in fibroblast spheroids. The endogenous level of stemness transcription factors ( OCT4, NANOG, SOX2, KLF4 and CMYC ) was higher in keratocytes than in fibroblasts. Immunofluorescence staining revealed Klf4, Nanog, Sox2, ABCG2 and Pax6 were positively stained in adherent 3D spheroids but weakly or negatively stained in adherent 2D cells. Furthermore, OCT4 , NANOG , SOX2 , KLF4 , HNK1 , ABCG2 and PAX6 gene expression was significantly higher in adherent 3D spheroids than in adherent 2D cells. Meanwhile, SOX2 , ABCG2 and PAX6 were more upregulated in adherent 3D spheroids than in suspended 3D spheroids. The RNA-seq analysis suggested that regulation of the actin cytoskeleton, TGFβ/BMP and HIF-1 signaling pathways induced changes in mechanotransduction, the mesenchymal-to-epithelial transition and hypoxia, which might be responsible for the effect of PDMS on facilitating reprogramming. In conclusion, compared to corneal fibroblasts, keratocytes were more susceptible to reprogramming due to higher levels of endogenous stemness transcription factors. Spheroid culture of keratocytes using PDMS had a positive impact on promoting the expression of some stem cell markers. PDMS, as a substrate to form spheroids, was better able to promote reprogramming than ULAPs. These results indicated that the physiological cells and culture conditions herein enhance reprogramming. Therefore, adherent spheroid culture of keratocytes using PDMS is a promising strategy to more safely promote reprogramming, suggesting its potential application for developing clinical implants in tissue engineering and regenerative medicine., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Li, Ding, Guo, Zhang, Wang, Sun, Zhang, Cui and Chen.)

Cell coculture strategies can promote angiogenesis within tissue engineering constructs. This study aimed to test the angiogenic potential of human umbilical vein endothelial cells (HUVEC) cocultured with gingiva-derived progenitor cells (GPC) as spheroids in a xeno-free environment. Human platelet lysate (HPL) was used as a cell culture supplement and as a hydrogel matrix (HPLG) for spheroid encapsulation. HUVEC and HUVEC + GPC (1:1 or 5:1) spheroids were encapsulated in various HPLG formulations. Angiogenesis was assessed via in vitro sprouting and in vivo chick chorioallantoic membrane (CAM) assays. HUVEC revealed characteristic in vitro sprouting in HPL/HPLG and this was significantly enhanced in cocultures with GPC ( p < 0.05). A trend for greater sprouting was observed in 5:1 vs 1:1 HUVEC + GPC spheroids and in certain HPLG formulations ( p > 0.05). Both HUVEC and HUVEC + GPC spheroids in HPLG revealed abundant and comparable neoangiogenesis in the CAM assay ( p > 0.05). Spheroid coculture of HUVEC + GPC in HPLG represents a promising strategy to promote angiogenesis., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2021 Shanbhag, Rashad, Nymark, Suliman, de Lange Davies, Stavropoulos, Bolstad and Mustafa.)

Three-dimensional (3D) cell culture in vitro has proven to be more physiologically relevant than two-dimensional (2D) culture of cell monolayers, thus more predictive in assessing efficacy and toxicity of compounds. There have been several 3D cell culture techniques developed, which include spheroid and multicellular tissue cultures. Cell spheroids have been generated from single or multiple cell types cultured in ultralow attachment (ULA) well plates and hanging droplet plates. In general, cell spheroids are formed in a relatively short period of culture, in the absence of extracellular matrices (ECMs), via gravity-driven self-aggregation, thus having limited ability to self-organization in layered structure. On the other hand, multicellular tissue cultures including miniature tissues derived from pluripotent stem cells and adult stem cells (a.k.a. 'organoids') and 3D bioprinted tissue constructs require biomimetic hydrogels or ECMs and show highly ordered structure due to spontaneous self-organization of cells during differentiation and maturation processes. In this short review article, we summarize traditional methods of spheroid and multicellular tissue cultures as well as their technical challenges, and introduce how droplet-based, miniature 3D bioprinting ('microarray 3D bioprinting') can be used to improve assay throughput and reproducibility for high-throughput, predictive screening of compounds. Several platforms including a micropillar chip and a 384-pillar plate developed to facilitate miniature spheroid and tissue cultures via microarray 3D bioprinting are introduced. We excluded microphysiological systems (MPSs) in this article although they are important tissue models to simulate multiorgan interactions., (© 2021 The Author(s). Published by Portland Press Limited on behalf of the Biochemical Society.)

Strategies for stem cell-based cardiac regeneration and repair are key issues for the ischemic heart disease (IHD) patients with chronic complications related to ischemic necrosis. Cardiac stem cells (CSCs) have demonstrated high therapeutic efficacy for IHD treatment owing to their specific cardiac-lineage commitment. The therapeutic potential of CSCs could be further enhanced by designing a cellular spheroid formulation. The spheroid culture condition of CSCs was optimized to ensure regulated size and minimal core necrosis in the spheroids. The CSC spheroids revealed mRNA profiles of the factors related to cardiac regeneration, angiogenesis, anti-inflammatory, and cardiomyocyte differentiation with a higher expression level than the CSCs. Intramyocardially delivered CSC spheroids in the rat IHD model resulted in a significant increase in retention rate by 1.82-fold (day 3) and 1.98-fold (day 14) compared to CSCs. Endothelial cell differentiation and neovascularization of the engrafted CSC spheroids were noted in the infarcted myocardium. CSC spheroids significantly promoted cardiac regeneration: i.e., decreased infarction and fibrotic area (11.22% and 4.18%) and increased left ventricle thickness (0.62 mm) compared to the untreated group. Cardiac performance was also improved by 2.04-fold and 1.44-fold increase in the ejection fraction and fractional shortening, respectively. Intramyocardial administration of CSC spheroids might serve as an advanced therapeutic modality with enhanced cell engraftment and regenerative abilities for cardiac repair after myocardial infarction., (Copyright © 2021 Elsevier B.V. All rights reserved.)

We present a spheroid trapping device, compatible with traditional tissue culture plates, to confine microtissues in a small area and allow suspension cultures to be treated like adherent cultures with minimal loss of spheroids due to aspiration. We also illustrate an automated morphology-independent procedure for cell recognition, segmentation, and a calcium spike detection technique for high-throughput analysis in 3D cultured tissue. Our cell recognition technique uses a maximum intensity projection of spatial-temporal data to create a binary mask, which delineates individual cell boundaries and extracts mean fluorescent data for each cell through a series of intensity thresholding and cluster labeling operations. The temporal data are subject to sorting for imaging artifacts, baseline correction, smoothing, and spike detection algorithms. We validated this procedure through analysis of calcium data from 2D and 3D SHSY-5Y cell cultures. Using this approach, we rapidly created regions of interest (ROIs) and extracted fluorescent intensity data from hundreds of cells in the field of view with superior data fidelity over hand-drawn ROIs even in dense (3D tissue) cell populations. We sorted data from cells with imaging artifacts (such as photo bleaching and dye saturation), classified nonfiring and firing cells, estimated the number of spikes in each cell, and documented the results, facilitating large-scale calcium imaging analysis in both 2D and 3D cultures. Since our recognition and segmentation technique is independent of morphology, our protocol provides a versatile platform for the analysis of large confocal calcium imaging data from neuronal cells, glial cells, and other cell types.

Background: Sweat glands (SGs) and hair follicles (HFs) are two important cutaneous appendages that play crucial roles in homeostatic maintenance and thermoregulation, and their interaction is involved in wound healing. SGs can be regenerated from mesenchymal stem cell-laden 3D bioprinted scaffolds, based on our previous studies, whereas regeneration of HFs could not be achieved in the same model. Due to the lack of an in vitro model, the underlying molecular mechanism of the interaction between SGs and HFs in regeneration could not be fully understood. The purpose of the present study was to establish an in vitro model of skin constructs with SGs and HFs and explore the interaction between these two appendages in regeneration., Methods: To investigate the interaction effects between SGs and HFs during their regeneration processes, a combined model was created by seeding HF spheroids on 3D printed SG scaffolds. The interaction between SG scaffolds and HF spheroids was detected using RNA expression and immunofluorescence staining. The effects of microenvironmental cues on SG and HF regeneration were analysed by altering seed cell types and plantar dermis homogenate in the scaffold., Results: According to this model, we overcame the difficulties in simultaneously inducing SG and HF regeneration and explored the interaction effects between SG scaffolds and HF spheroids. Surprisingly, HF spheroids promoted both SG and HF differentiation in SG scaffolds, while SG scaffolds promoted SG differentiation but had little effect on HF potency in HF spheroids. Specifically, microenvironmental factors (plantar dermis homogenate) in SG scaffolds effectively promoted SG and HF genesis in HF spheroids, no matter what the seed cell type in SG scaffolds was, and the promotion effects were persistent., Conclusions: Our approach elucidated a new model for SG and HF formation in vitro and provided an applicable platform to investigate the interaction between SGs and HFs in vitro . This platform might facilitate 3D skin constructs with multiple appendages and unveil the spatiotemporal molecular program of multiple appendage regeneration., (© The Author(s) 2021. Published by Oxford University Press.)

Apoptotic effects of secoisolariciresinol diglucoside (SDG) in 2D and 3D cultures of SW480 cells were investigated. 40-200 μM SDG was used and IC50 values were determined for three different time intervals as 24, 48, or 72 hr for further experiments. BrdU, TUNEL, AIF, and caspase-3 stainings were used. SDG inhibited cell proliferation almost half and half for all time intervals in 2D and 3D cultures and also, induced apoptosis. Apoptotic cell percentages in the control group for 24, 48, and 72 hr were 27.00%, 29.00%, and 28.00%, respectively, while in the SDG treatment group were 59.00%, 61.00%, and 62.00%, respectively. In the spheroid cell culture, apoptotic cell percentages in the control group for 24, 48, and 72 hr were 6.90%, 7.20%, and 7.10%, respectively, while in the SDG treatment group were 19.50%, 19.50%, and 20.70%, respectively. Caspase-3 and AIF antibodies were used to indicate caspase-dependent and -independent apoptotic pathways. Significant increases were seen in both AIF and caspase-3 stainings when compared to the control group but caspase-3 staining results were significantly greater when compared to the AIF staining at all time intervals (p < .05). To prove this, CASP3 gene expression was evaluated by RT-qPCR. Unlike staining results, there was no statistically significant change at 24 hr in 2D and 3D cultures. But, significant upregulation at 48 (2.32-fold in 2D and 2.46-fold in 3D) and 72 hr (5.04-fold in 2D and 6.45-fold in 3D) were seen. PRACTICAL APPLICATIONS: Colon cancer is one of the most prevalent cancer in the developed countries and its etiology is complex. Although the underlying mechanisms are mostly unknown, the link between diet and colon cancer is known and dietary habits can promote cancer or protect against it. In recent years, flaxseed is accepted as a significant functional food ingredient and feeding with it could help in to prevent cancer. Secoisolariciresinol diglucoside is a flaxseed lignan and is metabolized to mammalian lignans by the gut. In the present study, SDG was evaluated for its apoptotic effects in colon carcinoma cell line via monolayer and spheroid cultures using immunohistochemical and gene expression techniques. Findings of this study suggest that SDG may protect against cancers and in particularly against colon cancer and further investigations has to be carried out for detailed underlying mechanisms., (© 2021 Wiley Periodicals LLC.)

Metastatic castrate-resistant prostate cancer (CRPC) remains uncurable and novel therapies are needed to better treat patients. Aberrant Fibroblast Growth Factor Receptor (FGFR) signaling has been implicated in advanced prostate cancer (PCa), and FGFR1 is suggested to be a promising therapeutic target along with current androgen deprivation therapy. We established a novel in vitro 3D culture system to study endogenous FGFR signaling in a rare subpopulation of prostate cancer stem cells (CSCs) in the cell lines PC3, DU145, LNCaP, and the induced pluripotent iPS87 cell line. 3D-propagation of PCa cells generated spheroids with increased stemness markers ALDH7A1 and OCT4, while inhibition of FGFR signaling by BGJ398 or Dovitinib decreased cell survival and proliferation of 3D spheroids. The 3D spheroids exhibited altered expression of EMT markers associated with metastasis such as E-cadherin, vimentin and Snail, compared to 2D monolayer cells. TKI treatment did not result in significant changes of EMT markers, however, specific inhibition of FGFR signaling by BGJ398 showed more favorable molecular-level changes than treatment with the multi-RTK inhibitor Dovitinib. This study provides evidence for the first time that FGFR1 plays an essential role in the proliferation of PCa CSCs at a molecular and cellular level, and suggests that TKI targeting of FGFR signaling may be a promising strategy for AR-independent CRPC., Competing Interests: CONFLICTS OF INTEREST Authors have no conflicts of interest to declare., (Copyright: © 2021 Ko et al.)

Periodontitis is a progressive inflammation disease, the clinical management of which remains a challenge. The traditional management may control periodontal inflammation, but failed to regenerate functional periodontium. This review summarizes the most advancing regenerative techniques regarding stem cell culture and scaffold fabrication, such as cell sheeting, spheroid culture, electrospinning and 3D printing. The applications of different techniques manifest tremendous potential of regenerating the complete and functional periodontium. Albeit promising, new technologies have met with their own drawbacks such as insufficient vascularization and precision, which necessitate deeper modification. Thus, this review also points out the potential perspectives and methods aiming at their disadvantages, illuminating the directions of future researches to successful clinical scenarios., Competing Interests: The authors have no conflicts of interest related to this article., (© 2020 Association for Dental Sciences of the Republic of China. Publishing services by Elsevier B.V.)

In this paper, we developed a spheroid culture device that can trap a spheroid in the trapping site sandwiched by two extracellular matrix gels located at the upper and lower side of the spheroid. This device can form different biochemical gradients by applying target biochemicals separately in upper and lower channels, allowing us to study the angiogenic sprouting under various biochemical gradients in different directions. In the experiments, we confirmed the trapping of the spheroids and demonstrate the investigation on the direction and extent of angiogenic sprouts under unidirectional or bidirectional biochemical gradients. We believe our device can contribute to understanding the pathophysiological phenomena driven by chemical gradients, such as tissue development and tumor angiogenesis.

There is a growing body of evidence suggesting antitumor activity of statins. In metastasis and invasion of cancer the Epithelial-Mesenchymal Transition (EMT) of cancerous cells is an important process. Our goal was to understand the effect of Rosuvastatin on the EMT process in human prostate cancer cell line PC-3 cells in adherent 2 dimensional (2D) and spheroid 3 dimensional (3D) culture. PC-3 cells were cultured in adherence and/or spheroid culture system. The cells were treated with different concentrations of Rosuvastatin. After 96 h, the cell proliferation, viability, type and number of spheroids, the expression of E-Cadherin, Vimentin and Zeb-1 were analyzed. The results show that Rosuvastatin inhibit cell proliferation without significant cytotoxicity. The spheroid formation and spheroid sizes were inhibited by Rousavastatin in a dose dependent manner. In 2D culture, expression of the E-Cadherin was increased up to 2.0 fold in a dose dependent linear manner (R 2  = 0.89). Vimentin and Zeb-1 expressions were decreased up to 40 and 20% of untreated control cells expression level respectively, (R 2  = 0.99 and 0.92). In 3D system, the expression of E-Cadherin did not show a significant change, but Vimentin and Zeb-1 expressions were decreased up to 70 and 40% of untreated control cells expression level respectively in a dose dependent linear manner in comparison to 2D system (R 2  = 0.36 and 0.90). Our finding indicates that Rousavastatin inhibit cell proliferation and spheroid formation of PC-3 cells. This inhibition accompanies by inhibition of EMT markers. Therefor, this cholesterol lowering agent could probably have potential in the prevention and suppression of cancer in androgen dependent prostate cancer.

Spheroidal cancer cell cultures have been used to enrich cancer stem cells (CSC), which are thought to contribute to important clinical features of tumors. This study aimed to map the regulatory networks driven by circular RNAs (circRNAs) in CSC-enriched colorectal cancer (CRC) spheroid cells. The spheroid cells established from two CRC cell lines acquired stemness properties in pluripotency gene expression and multi-lineage differentiation capacity. Genome-wide sequencing identified 1503 and 636 circRNAs specific to the CRC parental and spheroid cells, respectively. In the CRC spheroids, algorithmic analyses unveiled a core network of mRNAs involved in modulating stemness-associated signaling pathways, driven by a circRNA-microRNA (miRNA)-mRNA axis. The two major circRNAs, hsa&#95;circ&#95;0066631 and hsa&#95;circ&#95;0082096, in this network were significantly up-regulated in expression levels in the spheroid cells. The two circRNAs were predicted to target and were experimentally shown to down-regulate miR-140-3p, miR-224, miR-382, miR-548c-3p and miR-579, confirming circRNA sponging of the targeted miRNAs. Furthermore, the affected miRNAs were demonstrated to inhibit degradation of six mRNA targets, viz. ACVR1C/ALK7, FZD3, IL6ST/GP130, SKIL/SNON, SMAD2 and WNT5, in the CRC spheroid cells. These mRNAs encode proteins that are reported to variously regulate the GP130/Stat, Activin/Nodal, TGF-β/SMAD or Wnt/β-catenin signaling pathways in controlling various aspects of CSC stemness. Using the CRC spheroid cell model, the novel circRNA-miRNA-mRNA axis mapped in this work forms the foundation for the elucidation of the molecular mechanisms of the complex cellular and biochemical processes that determine CSC stemness properties of cancer cells, and possibly for designing therapeutic strategies for CRC treatment by targeting CSC.

Testicular germ cell tumor (GCT) is the most common type of malignancy in young males. Patients with nonseminomatous GCT still have poor prognosis. To identify new therapeutic targets, we generated patient-derived cells (PDCs) and their xenograft (PDCX) models from 3 distinct GCT patients' specimens. The pathological features of GCT PDCs and PDCX tumors recapitulated those of nonseminomatous components exhibiting in the corresponding patients' specimens. Notably, stemness-related markers and hypoxia-related genes, including hypoxia inducible factor 1α (HIF1A) and neuritin 1 (NRN1), were abundantly expressed in three-dimensional spheroid cultures of GCT PDCs. We identified functional HIF1α response elements in the NRN1 promoter and defined that their transcriptional activities were substantially activated by hypoxia. HIF1α inhibition by siRNAs or an inhibitor, 2-methoxyestradiol, significantly suppressed NRN1 expression and decreased the in vitro and in vivo growth of PDC spheroids. Moreover, NRN1 knockdown efficiently suppressed PDC proliferation. These results suggest that HIF1α and NRN1 are potential diagnostic and therapeutic targets, and that 2-methoxyestradiol could be applied to clinical management of GCT. Overall, our GCT PDC and PDCX models would be useful as preclinical models for precision medicine targeting each patient., (Copyright © 2020 Elsevier B.V. All rights reserved.)

Gingiva has been identified as a minimally invasive source of multipotent progenitor cells (GPCs) for use in bone tissue engineering (BTE). To facilitate clinical translation, it is important to characterize GPCs in xeno-free cultures. Recent evidence indicates several advantages of three-dimensional (3D) spheroid cultures of mesenchymal stromal cells (MSCs) over conventional 2D monolayers. The present study aimed to characterize human GPCs in xeno-free 2D cultures, and to test their osteogenic potential in 3D cultures, in comparison to bone marrow MSCs (BMSCs). Primary GPCs and BMSCs were expanded in human platelet lysate (HPL) or fetal bovine serum (FBS) and characterized based on in vitro proliferation, immunophenotype and multi-lineage differentiation. Next, 3D spheroids of GPCs and BMSCs were formed via self-assembly and cultured in HPL. Expression of stemness- (SOX2, OCT4, NANOG) and osteogenesis-related markers (BMP2, RUNX2, OPN, OCN) was assessed at gene and protein levels in 3D and 2D cultures. The cytokine profile of 3D and 2D GPCs and BMSCs was assessed via a multiplex immunoassay. Monolayer GPCs in both HPL and FBS demonstrated a characteristic MSC-like immunophenotype and multi-lineage differentiation; osteogenic differentiation of GPCs was enhanced in HPL vs. FBS. CD271 + GPCs in HPL spontaneously acquired a neuronal phenotype and strongly expressed neuronal/glial markers. 3D spheroids of GPCs and BMSCs with high cell viability were formed in HPL media. Expression of stemness- and osteogenesis-related genes was significantly upregulated in 3D vs. 2D GPCs/BMSCs; the latter was independent of osteogenic induction. Synthesis of SOX2, BMP2 and OCN was confirmed via immunostaining, and in vitro mineralization via Alizarin red staining. Finally, secretion of several growth factors and chemokines was enhanced in GPC/BMSC spheroids, while that of pro-inflammatory cytokines was reduced, compared to monolayers. In summary, monolayer GPCs expanded in HPL demonstrate enhanced osteogenic differentiation potential, comparable to that of BMSCs. Xeno-free spheroid culture further enhances stemness- and osteogenesis-related gene expression, and cytokine secretion in GPCs, comparable to that of BMSCs., (Copyright © 2020 Shanbhag, Suliman, Bolstad, Stavropoulos and Mustafa.)

The expanded use of second-generation antiandrogens revolutionized the treatment landscape of progressed prostate cancer. However, resistances to these novel drugs are already the next obstacle to be solved. Various previous studies depicted an involvement of the enzyme AKR1C3 in the process of castration resistance as well as in the resistance to 2nd generation antiandrogens like enzalutamide. In our study, we examined the potential of natural AKR1C3 inhibitors in various prostate cancer cell lines and a three-dimensional co-culture spheroid model consisting of cancer cells and cancer-associated fibroblasts (CAFs) mimicking enzalutamide resistant prostate cancer. One of our compounds, named MF-15, expressed strong antineoplastic effects especially in cell culture models with significant enzalutamide resistance. Furthermore, MF-15 exhibited a strong effect on androgen receptor (AR) signaling, including significant inhibition of AR activity, downregulation of androgen-regulated genes, lower prostate specific antigen (PSA) production, and decreased AR and AKR1C3 expression, indicating a bi-functional effect. Even more important, we demonstrated a persisting inhibition of AR activity in the presence of AR-V7 and further showed that MF-15 non-competitively binds within the DNA binding domain of the AR. The data suggest MF-15 as useful drug to overcome enzalutamide resistance.

Although regenerative medicine products are at the forefront of scientific research, technological innovation, and clinical translation, their reproducibility and large-scale production are compromised by automation, monitoring, and standardization issues. To overcome these limitations, new technologies at software (e.g., algorithms and artificial intelligence models, combined with imaging software and machine learning techniques) and hardware (e.g., automated liquid handling, automated cell expansion bioreactor systems, automated colony-forming unit counting and characterization units, and scalable cell culture plates) level are under intense investigation. Automation, monitoring and standardization should be considered at the early stages of the developmental cycle of cell products to deliver more robust and effective therapies and treatment plans to the bedside, reducing healthcare expenditure and improving services and patient care., (Copyright © 2020 Doulgkeroglou, Di Nubila, Niessing, König, Schmitt, Damen, Szilvassy, Chang, Csontos, Louis, Kugelmeier, Ronfard, Bayon and Zeugolis.)

With the rise of obesity, diabetes, and other metabolic diseases, in vitro hepatic cell and tissue models play an essential role in the identification of active pharmaceutical ingredients. Up to now, three-dimensional (3D) culture models have rarely focused on hepatic glucose and lipid metabolism. In addition, primary human liver cells suffer from limited availability and interdonor difference for establishing reproducible models. Thus, in the current study, the most available human liver cancer cell line (HepG2) and primary hepatocytes from rats (rPH) were proposed to construct 3D spheroids using injectable fiber fragments with galactose grafts (gSF) as the substrate. rPH and HepG2 spheroids show strong cell-cell and cell-fiber fragment interactions to promote the cell viability, albumin, and urea syntheses. Compared with HepG2 spheroids, rPH spheroids indicate stronger glucose metabolism abilities in terms of glucose consumption, intracellular glycogen content, gluconeogenesis rate, and sensitivity to glucose modulator hormones like insulin and glucagon. On the other hand, HepG2 spheroids display strong lipid metabolism abilities in producing significantly higher levels of total cholesterol and triglyceride. Compared with those without fiber fragments, the gSF-supported 3D culture establishes effective models for in vitro glucose (rPH spheroids) and lipid metabolisms (HepG2 spheroids). The screening models are confirmed from the respective enzyme activities and gene expressions and show significantly higher sensitivity and clinically related responses to hypoglycemic and lipid-lowering drugs. Thus, the culture configuration demonstrates a predictable in vitro platform for defining glucose and lipid metabolism profiles and screening therapeutic agents for metabolism disorders like diabetes and obesity., (© 2020 John Wiley & Sons, Ltd.)

Background: Cell-based therapies have been studied for articular cartilage regeneration. Articular cartilage defects have little treatments because articular cartilage was limited regenerative capacity. Damaged articular cartilage is difficult to obtain a successful therapeutic effect. In additionally these articular cartilage defects often cause osteoarthritis. Chondrocyte implantation is a widely available therapy used for regeneration of articular cartilage because this tissue has poor repair capacity after injury. Human nasal septum-drived chondrocytes (hNCs) from the septum show greater proliferation ability and chondrogenic capacity than human articular chondrocytes (hACs), even across different donors with different ages. Moreover, the chondrogenic properties of hNCs can be maintained after extensive culture expansion., Methods: In this study, 2 dimensional (2D) monolayer cultured hNCs (hNCs-2D) and 3 dimensional (3D) spheroids cultured hNCs (hNCs-3D) were examined for chondrogenic capacity in vitro by PCR and immunofluorescence staining for chondrogenic marker, cell survival during cultured and for cartilage regeneration ability in vivo in a rat osteochondral defect model., Results: hNCs-3D showed higher viability and more uniform morphology than 3D spheroids cultured hACs (hACs-3D) in culture. hNCs-3D also showed greater expression levels of the chondrocyte-specific marker Type II collagen (COL2A1) and sex-determining region Y (SRY)-box 9 (SOX9) than hNCs-2D. hNCs-3D also expressed chondrogenic markers in collagen. Specially, in the osteochondral defect model, implantation of hNCs-3D led to greater chondrogenic repair of focal cartilage defects in rats than implantation of hNCs-2D., Conclusion: These data suggest that hNCs-3D are valuable therapeutic agents for repair and regeneration of cartilage defects.

The dermal papilla comprises mesenchymal cells in hair follicles, which play the main role in regulating hair growth. Maintaining the potential hair inductivity of dermal papilla cells (DPCs) and dermal sheath cells during cell culture is the main factor in in vitro morphogenesis and regeneration of hair follicles. Using common methods for the cultivation of human dermal papilla reduces the maintenance requirements of the inductive capacity of the dermal papilla and the expression of specific dermal papilla biomarkers. Optimizing culture conditions is therefore crucial for DPCs. Moreover, exosomes appear to play a key role in regulating the hair follicle growth through a paracrine mechanism and provide a functional method for treating hair loss. The present review investigated the biology of DPCs, the molecular and cell signaling mechanisms contributing to hair follicle growth in humans, the properties of the dermal papilla, and the effective techniques in maintaining hair inductivity in DPC cultures in humans as well as hair follicle bioengineering., (© 2020 S. Karger AG, Basel.)

Owing to the importance of stem cell culture systems in clinical applications, researchers have extensively studied them to optimize the culture conditions and increase efficiency of cell culture. A spheroid culture system provides a similar physicochemical environment in vivo by facilitating cell-cell and cell-matrix interaction to overcome the limitations of traditional monolayer cell culture. In suspension culture, aggregates of adjacent cells form a spheroid shape having wide utility in tumor and cancer research, therapeutic transplantation, drug screening, and clinical study, as well as organic culture. There are various spheroid culture methods such as hanging drop, gel embedding, magnetic levitation, and spinner culture. Lately, efforts are being made to apply the spheroid culture system to the study of drug delivery platforms and co-cultures, and to regulate differentiation and pluripotency. To study spheroid cell culture, various kinds of biomaterials are used as building forms of hydrogel, film, particle, and bead, depending upon the requirement. However, spheroid cell culture system has limitations such as hypoxia and necrosis in the spheroid core. In addition, studies should focus on methods to dissociate cells from spheroid into single cells.

Mesenchymal stem cells (MSCs) are multipotent progenitor cells capable of differentiating into adipocytic, osteogenic, chondrogenic, and myogenic lineages. There is growing evidence that MSCs home into the tumor microenvironment attracted by a variety of signals such as chemokines, growth factors, and cytokines. Tumor-homing stem cells may originate from bone marrow-derived MSCs (BMSCs) or adipose tissue-derived MSCs. Recent scientific data suggest that MSCs in combination with tumor cells can either promote or inhibit tumorigenic behavior. In head and neck squamous cell carcinoma (HNSCC), BMSCs are reported to be enriched with a potential negative role. Here, we evaluated the effect of BMSCs from 4 different donors in combination with 4 HNSCC cell lines in a 3-dimensional multicellular spheroid model. Heterogeneous combinations revealed an up-regulation of gene and protein expression of osteogenic markers runt-related transcription factor 2 (RUNX2) and alkaline phosphatase (ALP) together with a substantial secretion of matrix metalloproteinase 9. Moreover, heterogenous BMSC/tumor spheroids showed increased invasion compared with homogenous spheroids in a Boyden chamber invasion assay. Furthermore, inhibition of ALP resulted in a substantially decreased spreading of heterogeneous spheroids on laminin-rich matrix. In summary, our data suggest a prometastatic effect of BMSCs combined with HNSCC.-Wessely, A., Waltera, A., Reichert, T. E., Stöckl, S., Grässel, S., Bauer, R. J. Induction of ALP and MMP9 activity facilitates invasive behavior in heterogeneous human BMSC and HNSCC 3D-spheroids.

The therapeutic application of recombinant proteins is limited due to their inherent structural complexity. Additionally, screening of therapeutic potential of protein products requires an appropriate testing platform to achieve biological relevance. Fabrication of three dimensional cultures bridges the gap between in vitro based monolayer cultures and clinical applications. In this perspective, glioblastoma U-87 MG and breast cancer MCF7 spheroids were generated to assess the therapeutic prospect of recombinant PTEN protein. PTEN bound to silver nanoclusters was encapsulated within PEG coating, which resulted in fabrication of spherical nanocarriers named as PTEN-nanocomposites. Internalization of PTEN-nanocomposites in the spheroids was confirmed by confocal microscopy. Upon uptake, PTEN-nanocomposites led to modulation of cyclins and apoptosis gene regulators culminating in cell cycle arrest and reduced cell viability as confirmed by calcein-AM/PI dual staining and alamar blue assay. Further, combination of tamoxifen and PTEN-nanocomposites on U-87 MG spheroids resulted in two-fold reduction of drug dosage. The study revealed that the monolayer culture results translated to the 3D culture as well, however higher dose of the recombinant PTEN was required for the spheroid system. The anti-proliferative role of PTEN-nanocomposites in a complex 3D environment augments its biological implication and paves the way for recombinant PTEN based therapeutic applications.

Suspension spheroid cultures of anchorage-dependent cell types have been widely used in cancer and stem cell research, as well as for producing organoids. It is believed that the 3-dimensional spheroid presents cells with a more physiological microenvironment to grow so that they behave more like cells in vivo, which is lacking in conventional 2-dimensional monolayer cultures. Recently, it has been reported that cancer cells grown as spheroids could express stem cell-associated genes. Hence, it is investigated whether normal mouse and human fibroblasts cultured as spheroids could also be induced to express stem cell-associated genes. The transcriptomes of human fibroblasts cultured as a monolayer and spheroids are compared and analyzed using real-time RT-qPCR, RNA-sequencing, and bioinformatics. The results reveal that the spheroid transcriptome resemble somatic cells being reprogramed into stem cells, including the induced expression of stemness-associated genes, increased expression of mesenchymal-to-epithelial transition-associated genes, and decreased expression of epithelial-to-mesenchymal transition-associated genes. In this context, it is hypothesized that during the process of spheroid formation, matrix-cell signaling is lost in favor of cell-cell contact signaling and that this subsequently increases the activity of the PI3K/Akt pathway that then upregulates Tbdx3 and stemness-associated genes., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Cancer stem cells (CSCs) are aggressive subpopulations with increased stem-like properties. CSCs are usually resistant to most standard therapies and are responsible for tumor repropagation. Similar to normal stem cells, isolation of CSCs is challenging due to the lack of reliable markers. Antigen-based sorting of CSCs usually requires staining with multiple markers, making the experiments complicated, expensive, and sometimes unreliable. Here, we study the feasibility of using dielectrophoresis (DEP) for isolation of glioblastoma cells with increased stemness. We culture a glioblastoma cell line in the form of neurospheres as an in vitro model for glioblastoma stem cells. We demonstrate that spheroid forming cells have higher expression of stem cell marker, nestin. Next, we show that dielectric properties of neurospheres change as a result of changing culture conditions. Our results indicate that spheroid forming cells need higher voltages to experience the same DEP force magnitude compared to normal monolayer cultures of glioblastoma cell line. This study confirms the possibility of using DEP to isolate glioblastoma stem cells., (© 2019 WILEY-VCH Verlag GmbH & Co. KGaA, Weinheim.)

Purpose: To generate and characterize 3D spheroid suspension cultures from radical prostatectomy (RP) specimens as a versatile model system for organ-confined prostate cancer (PCa)., Methods: Cancerous tissue samples from RP specimens were excised by a uropathologist. Preparation of 3D spheroids was done by mechanical disintegration and limited enzymatic digestion followed by serial filtration through 100 μm- and 40 μm-cell strainers. Thereafter, spheroids were cultured in a modified stem cell medium and characterized by a live/dead assay, whole-spheroid immunohistochemistry (IHC; CK5, CK8, AMACR, PSA, Ki67, AR, αSMA, Vimentin, E-Cadherin) and PSA-measurements in culture medium. Furthermore, their response to pharmaceutical treatment with docetaxel, bicalutamide, enzalutamide and abiraterone was tested., Results: 173 RP cases were included. The median preoperative PSA-level was 16.12 ng/ml [range 0.99;345], the median Gleason score was 7b [6;10]. 64 cases were excluded due to low tumor content in frozen sections (43) or to insufficient spheroid formation (21). In the remaining 109 cases, spheroids formed successfully and stayed viable for up to several months. IHC analysis revealed AR-, CK8-, and AMACR-positivity in nearly all cases, while CK5-positive cells were detectable only occasionally as were α-SMA and Vimentin. E-Cadherin was positive in most cases. Furthermore, spheroids proved to be amenable to cryopreservation. While abiraterone had no effect and docetaxel only a moderate effect, spheroid viability was markedly reduced upon bicalutamide and enzalutamide treatment., Conclusions: Multicellular 3D spheroids can be generated from patient-derived RP tissue samples and serve as an innovative in vitro model of organ-confined PCa.

Hair loss, also known as alopecia, is a common dermatological condition of psychosocial significance; development of therapeutic candidates for the treatment of this condition is, hence, important. Silibinin, a secondary metabolite from Silybum marianum , is an effective antioxidant that also prevents various cutaneous problems. In this study, we have investigated the effect of silibinin on hair induction using three-dimensional (3D) cultured, human dermal papilla (DP) spheroids. Silibinin was found to significantly increase viability through AKT serine/threonine kinase (AKT) activation in 3D DP spheroids. This was correlated with an increase in the diameter of the 3D DP spheroids. The activation of the wingless and INT-1 (Wnt)/β-catenin signaling pathway, which is associated with hair growth induction in the DP, was evaluated using the T cell-specific transcription factor and lymphoid enhancer-binding factor (TCF/LEF) transcription factor reporter assay; results indicated significantly increased luciferase activity. In addition, we were able to demonstrate increased expression of the target genes, WNT5a and LEF1 , using quantitative real-time PCR assay. Lastly, significantly elevated expression of signature genes associated with hair induction was demonstrated in the 3D DP spheroids treated with silibinin. These results suggest that silibinin promotes proliferation and hair induction through the AKT and Wnt/β-catenin signaling pathways in 3D DP spheroids. Silibinin can be a potential candidate to promote hair proliferation.