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Thanapirom, Kessarin, Elisabetta Caon, Margarita Papatheodoridi, Luca Frenguelli, Walid Al-Akkad, Zhang Zhenzhen, Maria Giovanna Vilia, Massimo Pinzani, Giuseppe Mazza, and Krista Rombouts. "Optimization and Validation of a Novel Three-Dimensional Co-Culture System in Decellularized Human Liver Scaffold for the Study of Liver Fibrosis and Cancer." Cancers 13, no. 19 (September 30, 2021): 4936. http://dx.doi.org/10.3390/cancers13194936.
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The introduction of new preclinical models for in vitro drug discovery and testing based on 3D tissue-specific extracellular matrix (ECM) is very much awaited. This study was aimed at developing and validating a co-culture model using decellularized human liver 3D ECM scaffolds as a platform for anti-fibrotic and anti-cancer drug testing. Decellularized 3D scaffolds obtained from healthy and cirrhotic human livers were bioengineered with LX2 and HEPG2 as single and co-cultures for up to 13 days and validated as a new drug-testing platform. Pro-fibrogenic markers and cancer phenotypic gene/protein expression and secretion were differently affected when single and co-cultures were exposed to TGF-β1 with specific ECM-dependent effects. The anti-fibrotic efficacy of Sorafenib significantly reduced TGF-β1-induced pro-fibrogenic effects, which coincided with a downregulation of STAT3 phosphorylation. The anti-cancer efficacy of Regorafenib was significantly reduced in 3D bioengineered cells when compared to 2D cultures and dose-dependently associated with cell apoptosis by cleaved PARP-1 activation and P-STAT3 inhibition. Regorafenib reversed TGF-β1-induced P-STAT3 and SHP-1 through induction of epithelial mesenchymal marker E-cadherin and downregulation of vimentin protein expression in both co-cultures engrafting healthy and cirrhotic 3D scaffolds. In their complex, the results of the study suggest that this newly proposed 3D co-culture platform is able to reproduce the natural physio-pathological microenvironment and could be employed for anti-fibrotic and anti-HCC drug screening.
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Truzzi, Francesca, Camilla Tibaldi, Anne Whittaker, Silvia Dilloo, Enzo Spisni, and Giovanni Dinelli. "Pro-Inflammatory Effect of Gliadins and Glutenins Extracted from Different Wheat Cultivars on an In Vitro 3D Intestinal Epithelium Model." International Journal of Molecular Sciences 22, no. 1 (December 26, 2020): 172. http://dx.doi.org/10.3390/ijms22010172.
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There is a need to assess the relationship between improved rheological properties and the immunogenic potential of wheat proteins. The present study aimed to investigate the in vitro effects of total protein extracts from three modern and two landrace Triticum aestivum commercial flour mixes, with significant differences in gluten strength (GS), on cell lines. Cytotoxicity and innate immune responses induced by wheat proteins were investigated using Caco-2 monocultures, two dimensional (2D) Caco-2/U937 co-cultures, and three dimensional (3D) co-cultures simulating the intestinal mucosa with Caco-2 epithelial cells situated above an extra-cellular matrix containing U937 monocytes and L929 fibroblasts. Modern wheat proteins, with increased GS, significantly reduced Caco-2 cell proliferation and vitality in monoculture and 2D co-cultures than landrace proteins. Modern wheat proteins also augmented Caco-2 monolayer disruption and tight junction protein, occludin, redistribution in 3D co-cultures. Release of interleukin-8 into the cell medium and increased U937 monocyte migration in both 2D and 3D co-cultures were similarly apparent. Immuno-activation of migrating U937 cells was evidenced from cluster of differentiation 14 (CD14) staining and CD11b-related differentiation into macrophages. The modern wheat proteins, with gluten polymorphism relatedness and increased GS, were shown to be more cytotoxic and immunogenic than the landrace wheat proteins.
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Smithmyer, Megan E., Christopher C. Deng, Samantha E. Cassel, Paige J. LeValley, Brent S. Sumerlin, and April M. Kloxin. "Self-Healing Boronic Acid-Based Hydrogels for 3D Co-cultures." ACS Macro Letters 7, no. 9 (August 31, 2018): 1105–10. http://dx.doi.org/10.1021/acsmacrolett.8b00462.
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Soriano, Luis, Tehreem Khalid, Fergal J. O'Brien, Cian O'Leary, and Sally-Ann Cryan. "A Tissue-Engineered Tracheobronchial In Vitro Co-Culture Model for Determining Epithelial Toxicological and Inflammatory Responses." Biomedicines 9, no. 6 (June 2, 2021): 631. http://dx.doi.org/10.3390/biomedicines9060631.
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Translation of novel inhalable therapies for respiratory diseases is hampered due to the lack of in vitro cell models that reflect the complexity of native tissue, resulting in many novel drugs and formulations failing to progress beyond preclinical assessments. The development of physiologically-representative tracheobronchial tissue analogues has the potential to improve the translation of new treatments by more accurately reflecting in vivo respiratory pharmacological and toxicological responses. Herein, advanced tissue-engineered collagen hyaluronic acid bilayered scaffolds (CHyA-B) previously developed within our group were used to evaluate bacterial and drug-induced toxicity and inflammation for the first time. Calu-3 bronchial epithelial cells and Wi38 lung fibroblasts were grown on either CHyA-B scaffolds (3D) or Transwell® inserts (2D) under air liquid interface (ALI) conditions. Toxicological and inflammatory responses from epithelial monocultures and co-cultures grown in 2D or 3D were compared, using lipopolysaccharide (LPS) and bleomycin challenges to induce bacterial and drug responses in vitro. The 3D in vitro model exhibited significant epithelial barrier formation that was maintained upon introduction of co-culture conditions. Barrier integrity showed differential recovery in CHyA-B and Transwell® epithelial cultures. Basolateral secretion of pro-inflammatory cytokines to bacterial challenge was found to be higher from cells grown in 3D compared to 2D. In addition, higher cytotoxicity and increased basolateral levels of cytokines were detected when epithelial cultures grown in 3D were challenged with bleomycin. CHyA-B scaffolds support the growth and differentiation of bronchial epithelial cells in a 3D co-culture model with different transepithelial resistance in comparison to the same co-cultures grown on Transwell® inserts. Epithelial cultures in an extracellular matrix like environment show distinct responses in cytokine release and metabolic activity compared to 2D polarised models, which better mimic in vivo response to toxic and inflammatory stimuli offering an innovative in vitro platform for respiratory drug development.
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Ruoß, Marc, Vanessa Kieber, Silas Rebholz, Caren Linnemann, Helen Rinderknecht, Victor Häussling, Marina Häcker, Leon H. H. Olde Damink, Sabrina Ehnert, and Andreas K. Nussler. "Cell-Type-Specific Quantification of a Scaffold-Based 3D Liver Co-Culture." Methods and Protocols 3, no. 1 (December 23, 2019): 1. http://dx.doi.org/10.3390/mps3010001.
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In order to increase the metabolic activity of human hepatocytes and liver cancer cell lines, many approaches have been reported in recent years. The metabolic activity could be increased mainly by cultivating the cells in 3D systems or co-cultures (with other cell lines). However, if the system becomes more complex, it gets more difficult to quantify the number of cells (e.g., on a 3D matrix). Until now, it has been impossible to quantify different cell types individually in 3D co-culture systems. Therefore, we developed a PCR-based method that allows the quantification of HepG2 cells and 3T3-J2 cells separately in a 3D scaffold culture. Moreover, our results show that this method allows better comparability between 2D and 3D cultures in comparison to the often-used approaches based on metabolic activity measurements, such as the conversion of resazurin.
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Rausch, Magdalena, Léa Blanc, Olga De Souza Silva, Olivier Dormond, Arjan W. Griffioen, and Patrycja Nowak-Sliwinska. "Characterization of Renal Cell Carcinoma Heterotypic 3D Co-Cultures with Immune Cell Subsets." Cancers 13, no. 11 (May 22, 2021): 2551. http://dx.doi.org/10.3390/cancers13112551.
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Two-dimensional cell culture-based platforms are easy and reproducible, however, they do not resemble the heterotypic cell-cell interactions or the complex tumor microenvironment. These parameters influence the treatment response and the cancer cell fate. Platforms to study the efficacy of anti-cancer treatments and their impact on the tumor microenvironment are currently being developed. In this study, we established robust, reproducible, and easy-to-use short-term spheroid cultures to mimic clear cell renal cell carcinoma (ccRCC). These 3D co-cultures included human endothelial cells, fibroblasts, immune cell subsets, and ccRCC cell lines, both parental and sunitinib-resistant. During spheroid formation, cells induce the production and secretion of the extracellular matrix. We monitored immune cell infiltration, surface protein expression, and the response to a treatment showing that the immune cells infiltrated the spheroid co-cultures within 6 h. Treatment with an optimized drug combination or the small molecule-based targeted drug sunitinib increased immune cell infiltration significantly. Assessing the therapeutic potential of this drug combination in this platform, we revealed that the expression of PD-L1 increased in 3D co-cultures. The cost- and time-effective establishment of our 3D co-culture model and its application as a pre-clinical drug screening platform can facilitate the treatment validation and clinical translation.
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Martinez-Pacheco, Sarai, and Lorraine O’Driscoll. "Pre-Clinical In Vitro Models Used in Cancer Research: Results of a Worldwide Survey." Cancers 13, no. 23 (November 30, 2021): 6033. http://dx.doi.org/10.3390/cancers13236033.
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To develop and subsequently get cancer researchers to use organotypic three-dimensional (3D) models that can recapitulate the complexity of human in vivo tumors in an in vitro setting, it is important to establish what in vitro model(s) researchers are currently using and the reasons why. Thus, we developed a survey on this topic, obtained ethics approval, and circulated it throughout the world. The survey was completed by 101 researchers, across all career stages, in academia, clinical or industry settings. It included 40 questions, many with multiple options. Respondents reported on their field of cancer research; type of cancers studied; use of two-dimensional (2D)/monolayer, 2.5D and/or 3D cultures; if using co-cultures, the cell types(s) they co-culture; if using 3D cultures, whether these involve culturing the cells in a particular way to generate spheroids, or if they use additional supports/scaffolds; techniques used to analyze the 2D/2.5D/3D; and their downstream applications. Most researchers (>66%) only use 2D cultures, mainly due to lack of experience and costs. Despite most cancer researchers currently not using the 3D format, >80% recognize their importance and would like to progress to using 3D models. This suggests an urgent need to standardize reliable, robust, reproducible methods for establishing cost-effective 3D cell culture models and their subsequent characterization.
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Meretoja, Ville V., Rebecca L. Dahlin, Sarah Wright, F. Kurtis Kasper, and Antonios G. Mikos. "Articular Chondrocyte Redifferentiation in 3D Co-cultures with Mesenchymal Stem Cells." Tissue Engineering Part C: Methods 20, no. 6 (June 2014): 514–23. http://dx.doi.org/10.1089/ten.tec.2013.0532.
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Nuñez-Olvera, Stephanie I., Lorena Aguilar-Arnal, Mireya Cisneros-Villanueva, Alfredo Hidalgo-Miranda, Laurence A. Marchat, Yarely M. Salinas-Vera, Rosalio Ramos-Payán, et al. "Breast Cancer Cells Reprogram the Oncogenic lncRNAs/mRNAs Coexpression Networks in Three-Dimensional Microenvironment." Cells 11, no. 21 (November 1, 2022): 3458. http://dx.doi.org/10.3390/cells11213458.
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Organotypic three-dimensional (3D) cell cultures more accurately mimic the characteristics of solid tumors in vivo in comparison with traditional two-dimensional (2D) monolayer cell models. Currently, studies on the regulation of long non-coding RNAs (lncRNAs) have not been explored in breast cancer cells cultured in 3D microenvironments. In the present research, we studied the expression and potential roles of lncRNAs in estrogen receptor-positive luminal B subtype BT-474 breast cancer cells grown over extracellular matrix proteins-enriched 3D cultures. Global expression profiling using DNA microarrays identifies 290 upregulated and 183 downregulated lncRNAs in 3D cultures relative to 2D condition. Using a co-expression analysis approach of lncRNAs and mRNAs pairs expressed in the same experimental conditions, we identify hundreds of regulatory axes modulating genes involved in cancer hallmarks, such as responses to estrogens, cell proliferation, hypoxia, apical junctions, and resistance to endocrine therapy. In addition, we identified 102 lncRNAs/mRNA correlations in 3D cultures, which were similar to those reported in TCGA datasets obtained from luminal B breast cancer patients. Interestingly, we also found a set of mRNAs transcripts co-expressed with LINC00847 and CTD-2566J3.1 lncRNAs, which were predictors of pathologic complete response and overall survival. Finally, both LINC00847 and CTD -2566J3.1 were co-expressed with essential genes for cancer genetic dependencies, such as FOXA1 y GINS2. Our experimental and predictive findings show that co-expressed lncRNAs/mRNAs pairs exhibit a high degree of similarity with those found in luminal B breast cancer patients, suggesting that they could be adequate pre-clinical tools to identify not only biomarkers related to endocrine therapy response and PCR, but to understand the biological behavior of cancer cells in 3D microenvironments.
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Kaushik, Garima, Bhavna Verma, and Amy Wesa. "88 Development of a 3D organoid autologous TIL co-culture platform for high throughput immuno-oncology studies." Journal for ImmunoTherapy of Cancer 8, Suppl 3 (November 2020): A98. http://dx.doi.org/10.1136/jitc-2020-sitc2020.0088.
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BackgroundThe preclinical screening of immune-modulatory therapies suffers from the absence of models that recapitulate in vivo heterogeneous tumor microenvironment (TME). 3D tumor organoid cultures provide a model that closely mimics in situ tumor architecture and is being aggressively used to evaluate therapeutic efficacy ex vivo. A vastly heterogenous TME impacts patient treatment response, and there is a dearth of human tumor models (2D or 3D), that mimic in vivo diversity of TME, including infiltrating immune populations. 3D organoid cultures typically contain neoplastic epithelium; however, they fall short in representing tumor to tumor-infiltrating lymphocytes (TILs) interactions, limiting their ability to generate a clinically relevant response to immunotherapeutics. Addition of immune cells from unrelated donors to organoids can simulate that microenvironment but is complicated by T cell alloreactivity. Here we describe 3D patient-derived xenograft organoid (PDXO) co-cultures with matching autologous human TILs to recapitulate the tumor-specific immune response, leveraging confocal high content analysis and luminex multiplex assays. This platform allows the evaluation and high throughput screening of novel immune targeting agents to determine impacts on patient-derived T cell function, T cell infiltration, and tumor cytotoxicity.MethodsSurgical resections from patients were used to generate patient-derived xenografts and tumor-infiltrating lymphocytes in parallel. PDX were resected and digested to establish PDXO. TILs and organoids from the same patient were fluorescent labeled and cultured together for four days to evaluate tumor infiltration and drug cytotoxicity in 3D cultures. CellInsight CX7 high content imaging platform was used to trace TILs and cancer cells and evaluate T cell infiltration and tumor cell killing in the presence and absence of immuno-modulatory therapies.ResultsPDXO were established to mimic in vivo tumor biology. Tumor-specific TILs were successfully expanded and characterized by flow cytometry. Co-culture resulted in TIL infiltration in organoids from day one in culture and increased over four days. Cytotoxicity and TIL infiltration were quantified using fluorescent dyes via high throughput imaging platform. Significantly enhanced TIL infiltration was observed in autologous co-cultures compared to non-autologous co-cultures. The established unique autologous PDXO immune organoid co-cultures could be used as an improved simulation of the modulatory activity of therapeutic agents in patient-specific T cells against their own tumors.ConclusionsPatient autologous TILs – PDXO co-culture platform is an advanced model for evaluating IO therapeutics with the tumor-specific immune microenvironment. The platform provides an opportunity for precision medicine and high throughput drug screening of immuno-modulatory therapies.Ethics ApprovalThe study was approved by Champions Oncology’s Institutional Animal Care and Use Committee (IACUC).
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Dozzo, Annachiara, Krishnakumar Chullipalliyalil, Michael McAuliffe, Caitriona M. O’Driscoll, and Katie B. Ryan. "Nano-Hydroxyapatite/PLGA Mixed Scaffolds as a Tool for Drug Development and to Study Metastatic Prostate Cancer in the Bone." Pharmaceutics 15, no. 1 (January 11, 2023): 242. http://dx.doi.org/10.3390/pharmaceutics15010242.
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(1) Background: Three-dimensional (3D) in vitro, biorelevant culture models that recapitulate cancer progression can help elucidate physio-pathological disease cues and enhance the screening of more effective therapies. Insufficient research has been conducted to generate in vitro 3D models to replicate the spread of prostate cancer to the bone, a key metastatic site of the disease, and to understand the interplay between the key cell players. In this study, we aim to investigate PLGA and nano-hydroxyapatite (nHA)/PLGA mixed scaffolds as a predictive preclinical tool to study metastatic prostate cancer (mPC) in the bone and reduce the gap that exists with traditional 2D cultures. (2) Methods: nHA/PLGA mixed scaffolds were produced by electrospraying, compacting, and foaming PLGA polymer microparticles, +/− nano-hydroxyapatite (nHA), and a salt porogen to produce 3D, porous scaffolds. Physicochemical scaffold characterisation together with an evaluation of osteoblastic (hFOB 1.19) and mPC (PC-3) cell behaviour (RT-qPCR, viability, and differentiation) in mono- and co-culture, was undertaken. (3) Results: The results show that the addition of nHA, particularly at the higher-level impacted scaffolds in terms of mechanical and degradation behaviour. The nHA 4 mg resulted in weaker scaffolds, but cell viability increased. Qualitatively, fluorescent imaging of cultures showed an increase in PC-3 cells compared to osteoblasts despite lower initial PC-3 seeding densities. Osteoblast monocultures, in general, caused an upregulation (or at least equivalent to controls) in gene production, which was highest in plain scaffolds and decreased with increases in nHA. Additionally, the genes were downregulated in PC3 and co-cultures. Further, drug toxicity tests demonstrated a significant effect in 2D and 3D co-cultures. (4) Conclusions: The results demonstrate that culture conditions and environment (2D versus 3D, monoculture versus co-culture) and scaffold composition all impact cell behaviour and model development.
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Dudman, Joseph, Ana Marina Ferreira, Piergiorgio Gentile, Xiao Wang, and Kenneth Dalgarno. "Microvalve Bioprinting of MSC-Chondrocyte Co-Cultures." Cells 10, no. 12 (November 27, 2021): 3329. http://dx.doi.org/10.3390/cells10123329.
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Recent improvements within the fields of high-throughput screening and 3D tissue culture have provided the possibility of developing in vitro micro-tissue models that can be used to study diseases and screen potential new therapies. This paper reports a proof-of-concept study on the use of microvalve-based bioprinting to create laminar MSC-chondrocyte co-cultures to investigate whether the use of MSCs in ACI procedures would stimulate enhanced ECM production by chondrocytes. Microvalve-based bioprinting uses small-scale solenoid valves (microvalves) to deposit cells suspended in media in a consistent and repeatable manner. In this case, MSCs and chondrocytes have been sequentially printed into an insert-based transwell system in order to create a laminar co-culture, with variations in the ratios of the cell types used to investigate the potential for MSCs to stimulate ECM production. Histological and indirect immunofluorescence staining revealed the formation of dense tissue structures within the chondrocyte and MSC-chondrocyte cell co-cultures, alongside the establishment of a proliferative region at the base of the tissue. No stimulatory or inhibitory effect in terms of ECM production was observed through the introduction of MSCs, although the potential for an immunomodulatory benefit remains. This study, therefore, provides a novel method to enable the scalable production of therapeutically relevant micro-tissue models that can be used for in vitro research to optimise ACI procedures.
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von Tobel, Jenny Sandström, Igor Charvet, Sabrina Pagano, Nicolas Vuilleumier, Luc Stoppini, and Florianne Monnet-Tschudi. "Development of 3D human stem cells-microglia co-cultures for neurotoxicology studies." Toxicology Letters 211 (June 2012): S155. http://dx.doi.org/10.1016/j.toxlet.2012.03.564.
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van den Brand, A. D., E. Rubinstein, P. C. de Jong, M. van den Berg, and M. B. M. van Duursen. "Primary endometrial 3D co-cultures: A comparison between human and rat endometrium." Journal of Steroid Biochemistry and Molecular Biology 194 (November 2019): 105458. http://dx.doi.org/10.1016/j.jsbmb.2019.105458.
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Costa, Elisabete C., Vítor M. Gaspar, Paula Coutinho, and Ilídio J. Correia. "Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models." Biotechnology and Bioengineering 111, no. 8 (February 25, 2014): 1672–85. http://dx.doi.org/10.1002/bit.25210.
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Sun, Lin, Sara T. Parker, Daisuke Syoji, Xiuli Wang, Jennifer A. Lewis, and David L. Kaplan. "Direct-Write Assembly of 3D Silk/Hydroxyapatite Scaffolds for Bone Co-Cultures." Advanced Healthcare Materials 1, no. 6 (May 29, 2012): 729–35. http://dx.doi.org/10.1002/adhm.201200057.
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Engelmann, Luca, Julia Thierauf, Natalia Koerich Laureano, Hans-Juergen Stark, Elena-Sophie Prigge, Dominik Horn, Kolja Freier, et al. "Organotypic Co-Cultures as a Novel 3D Model for Head and Neck Squamous Cell Carcinoma." Cancers 12, no. 8 (August 18, 2020): 2330. http://dx.doi.org/10.3390/cancers12082330.
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Background: Head and neck squamous cell carcinomas (HNSCC) are phenotypically and molecularly heterogeneous and frequently develop therapy resistance. Reliable patient-derived 3D tumor models are urgently needed to further study the complex pathogenesis of these tumors and to overcome treatment failure. Methods: We developed a three-dimensional organotypic co-culture (3D-OTC) model for HNSCC that maintains the architecture and cell composition of the individual tumor. A dermal equivalent (DE), composed of healthy human-derived fibroblasts and viscose fibers, served as a scaffold for the patient sample. DEs were co-cultivated with 13 vital HNSCC explants (non-human papillomavirus (HPV) driven, n = 7; HPV-driven, n = 6). Fractionated irradiation was applied to 5 samples (non-HPV-driven, n = 2; HPV-driven n = 3). To evaluate expression of ki-67, cleaved caspase-3, pan-cytokeratin, p16INK4a, CD45, ∝smooth muscle actin and vimentin over time, immunohistochemistry and immunofluorescence staining were performed Patient checkup data were collected for up to 32 months after first diagnosis. Results: All non-HPV-driven 3D-OTCs encompassed proliferative cancer cells during cultivation for up to 21 days. Proliferation indices of primaries and 3D-OTCs were comparable and consistent over time. Overall, tumor explants displayed heterogeneous growth patterns (i.e., invasive, expansive, silent). Cancer-associated fibroblasts and leukocytes could be detected for up to 21 days. HPV DNA was detectable in both primary and 3D-OTCs (day 14) of HPV-driven tumors. However, p16INK4a expression levels were varying. Morphological alterations and radioresistant tumor cells were detected in 3D-OTC after fractionated irradiation in HPV-driven and non-driven samples. Conclusions: Our 3D-OTC model for HNSCC supports cancer cell survival and proliferation in their original microenvironment. The model enables investigation of invasive cancer growth and might, in the future, serve as a platform to perform sensitivity testing upon treatment to predict therapy response.
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Han, Yilin, Marianne King, Evgenii Tikhomirov, Povilas Barasa, Cleide Dos Santos Souza, Jonas Lindh, Daiva Baltriukiene, et al. "Towards 3D Bioprinted Spinal Cord Organoids." International Journal of Molecular Sciences 23, no. 10 (May 21, 2022): 5788. http://dx.doi.org/10.3390/ijms23105788.
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Three-dimensional (3D) cultures, so-called organoids, have emerged as an attractive tool for disease modeling and therapeutic innovations. Here, we aim to determine if boundary cap neural crest stem cells (BC) can survive and differentiate in gelatin-based 3D bioprinted bioink scaffolds in order to establish an enabling technology for the fabrication of spinal cord organoids on a chip. BC previously demonstrated the ability to support survival and differentiation of co-implanted or co-cultured cells and supported motor neuron survival in excitotoxically challenged spinal cord slice cultures. We tested different combinations of bioink and cross-linked material, analyzed the survival of BC on the surface and inside the scaffolds, and then tested if human iPSC-derived neural cells (motor neuron precursors and astrocytes) can be printed with the same protocol, which was developed for BC. We showed that this protocol is applicable for human cells. Neural differentiation was more prominent in the peripheral compared to central parts of the printed construct, presumably because of easier access to differentiation-promoting factors in the medium. These findings show that the gelatin-based and enzymatically cross-linked hydrogel is a suitable bioink for building a multicellular, bioprinted spinal cord organoid, but that further measures are still required to achieve uniform neural differentiation.
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Quail, Daniela F., Tamara J. Maciel, Kem Rogers, and Lynne M. Postovit. "A Unique 3D In Vitro Cellular Invasion Assay." Journal of Biomolecular Screening 17, no. 8 (June 15, 2012): 1088–95. http://dx.doi.org/10.1177/1087057112449863.
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Three-dimensional (3D) cell culture techniques using a bioreactor have been used to co-culture various breast cancer cell lines. Comparisons between 3D co-cultures containing different proportions of breast cancer cell lines have been made with respect to cluster size, cell surface marker distribution, and Ki67 expression. Furthermore, an observed difference in invasion through collagen between co-cultures has been briefly reported. However, these assays have not yet been developed into a quantifiable methodology to assess the effects of drugs and/or microenvironments on cellular invasion. From a cancer perspective, two important aspects of cellular invasion that are often left out of in vitro assays are considerations about the 3D structural heterogeneity of the primary tumor and the ability of cells to migrate in all directions. Accordingly, we have taken advantage of the methodology previously described for 3D cell culture techniques and have developed a 3D invasion assay using cell clusters that can be used to assess the effects of different drugs and treatment conditions on cancer cell invasion. We also describe a novel whole-mount technique that permits fluorescence-based immunolocalization of proteins through the entire tumorsphere, without the need for sectioning. Our assay provides a simple, inexpensive, and physiologically relevant context to study cellular invasion in vitro, in a way that recapitulates an in vivo milieu.
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Ren, Gang, Xunzhen Zheng, Vandana Sharma, Joshua Letson, Andrea L. Nestor-Kalinoski, and Saori Furuta. "Loss of Nitric Oxide Induces Fibrogenic Response in Organotypic 3D Co-Culture of Mammary Epithelia and Fibroblasts—An Indicator for Breast Carcinogenesis." Cancers 13, no. 11 (June 5, 2021): 2815. http://dx.doi.org/10.3390/cancers13112815.
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Excessive myofibroblast activation, which leads to dysregulated collagen deposition and the stiffening of the extracellular matrix (ECM), plays pivotal roles in cancer initiation and progression. Cumulative evidence attests to the cancer-causing effects of a number of fibrogenic factors found in the environment, diseases and drugs. While identifying such factors largely depends on epidemiological studies, it would be of great importance to develop a robust in vitro method to demonstrate the causal relationship between fibrosis and cancer. Here, we tested whether our recently developed organotypic three-dimensional (3D) co-culture would be suitable for that purpose. This co-culture system utilizes the discontinuous ECM to separately culture mammary epithelia and fibroblasts in the discrete matrices to model the complexity of the mammary gland. We observed that pharmaceutical deprivation of nitric oxide (NO) in 3D co-cultures induced myofibroblast differentiation of the stroma as well as the occurrence of epithelial–mesenchymal transition (EMT) of the parenchyma. Such in vitro response to NO deprivation was unique to co-cultures and closely mimicked the phenotype of NO-depleted mammary glands exhibiting stromal desmoplasia and precancerous lesions undergoing EMT. These results suggest that this novel 3D co-culture system could be utilized in the deep mechanistic studies of the linkage between fibrosis and cancer.
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Adamik, Juraj, Yerneni Saigopalakrishna, Sree Pulugulla, Quanhong Sun, Philip E. Auron, Phil G. Campbell, and Deborah Lynn Galson. "Epigenetic Targeting of the Myeloma-Bone Microenvironment in 3D." Blood 132, Supplement 1 (November 29, 2018): 246. http://dx.doi.org/10.1182/blood-2018-99-119649.
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Abstract EZH2, the methyltransferase subunit of Polycomb Repressive Complex 2 (PRC2), catalyzes H3K27me3 histone modifications and epigenetically regulates genes involved in cellular pluripotency and differentiation. EZH2 plays a role in myeloma (MM) cell proliferation, survival, stemness and its elevated expression correlates with poor prognosis in MM patients. We have previously shown that EZH2 plays a critical role in preventing osteoblast differentiation of myeloma-exposed bone marrow stromal cells (BMSCs). Here we show that GSK126 blocks MM-induced hyperactivation of osteoclast precursors (OCLp). RNA-seq profiling revealed that inhibition of EZH2 prevented RANKL-induced repression of genes associated with bivalent and/or H3K27me3 promoter signatures including OCL inhibitory factors MafB, Irf8, Bcl6b and Arg1. In contrast, we found that OCLp expansion in MM1.S-conditioned media induced significant gene expression changes, which correlated with TNF and IKK signaling, inflammatory responses and CXC-chemokine receptor pathways. Several classes of small molecule EZH2 inhibitors exhibited anti-MM effects, but their efficacy has been primarily studied in 2-dimensional (2D) cell culture systems or subcutaneous MM-tumor models in vivo. Therefore, we evaluated the effectiveness of GSK126 in the context of the bone microenvironment in a novel 3D model of MM co-cultures (3D-MM). We combined basement membrane extract (BME) hydrogels with devitalized bone slices to mimic the 3D setting of MM with the OCL-resorbing endosteal surface. This enabled us to test GSK126 alone or in combination with bortezomib simultaneously on MM survival and OCLp differentiation and resorption. Differentiating OCLp did not protect MM cells from GSK126 anti-MM effects, nor did the MM cells prevent GSK126 from blocking OCL differentiation. However, mature OCL added to 3D-MM co-cultures increased the IC50 MM inhibition dose of both bortezomib and GSK126 by 40% and 50%, respectively. Further their synergy on MM cells was reduced by 70%. 3D-MM co-cultures with total bone marrows harvested from mice of different ages (1-12 months old) showed selective protection from GSK126, but not bortezomib, on MM viability. Furthermore, the resistance to GSK126 was age-dependent. Cultures of bone marrows from older mice exhibited enhanced protection of MM cells from GSK126 as compared to younger marrows. Using confocal microscopy, we found that in addition to soluble factors, physical interaction between MM and bone marrow cells reduced the effectiveness of the epigenetic drug targeting against MM. Depletion of the BMSC population from the total bone marrows using CD45+ selection before establishing the 3D MM co-cultures resulted in diminished protection of MM cell survival from GSK126. Consistent with this, addition of both primary murine and MM-patient derived BMSCs to MM cultures significantly protected MM cells from EZH2 inhibition. In addition to cell-cell contacts, the pro-survival factor IL6 released by mature OCL and BMSCs, has been implicated in mediating chemo-resistance of MM cells. In agreement with this, addition of soluble IL6 to MM-3D cultures significantly protected MM cells from GSK126 inhibition. Here we show that various cell compartments of the bone microenvironment exhibit differential and drug-specific protection for MM cells from EZH2 inhibition. In addition to direct bone marrow-MM cell interactions, soluble IL6 also exhibits resistance to GSK126. Our novel 3D-MM system enables us to rapidly screen drug combinations, and simultaneously evaluate the influence of bone-microenvironmental interactions on MM drug resistance and bone marrow cell responses to the drugs. Figure. Figure. Disclosures No relevant conflicts of interest to declare.
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Sebők, Csilla, Patrik Tráj, Júlia Vörösházi, Máté Mackei, Márton Papp, Péter Gálfi, Zsuzsanna Neogrády, and Gábor Mátis. "Two Sides to Every Question: Attempts to Activate Chicken Innate Immunity in 2D and 3D Hepatic Cell Cultures." Cells 10, no. 8 (July 27, 2021): 1910. http://dx.doi.org/10.3390/cells10081910.
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The liver with resident tissue macrophages is the site of vivid innate immunity, activated also by pathogen-associated molecular patterns (PAMPs) leaking through the intestinal barrier. As gut-derived inflammatory diseases are of outstanding importance in broiler chickens, the present study aimed to establish a proper hepatic inflammatory model by comparing the action of different PAMPs from poultry pathogens on chicken 2D and 3D primary hepatocyte—non-parenchymal cell co-cultures, the latter newly developed with a magnetic bioprinting method. The cultures were challenged by the bacterial endotoxins lipopolysaccharide (LPS) from Escherichia coli, lipoteichoic acid (LTA) from Staphylococcus aureus and by enterotoxin (ETxB) from Escherichia coli, Salmonella Typhimurium derived flagellin, phorbol myristate acetate (PMA) as a model proinflammatory agent and polyinosinic polycytidylic acid (poly I:C) for mimicking viral RNA exposure. Cellular metabolic activity was assessed with the CCK-8 test, membrane damage was monitored with the lactate dehydrogenase (LDH) leakage assay and interleukin-6 and -8 (Il-6 and -8) concentrations were measured in cell culture medium with a chicken specific ELISA. Both LPS and LTA increased the metabolic activity of the 3D cultures, concomitantly decreasing the LDH leakage, while in 2D cultures ETxB stimulated, PMA and poly I:C depressed the metabolic activity. Based on the moderately increased extracellular LDH activity, LTA seemed to diminish cell membrane integrity in 2D and poly I:C in both cell culture models. The applied endotoxins remarkably reduced the IL-8 release of 3D cultured cells, suggesting the effective metabolic adaptation and the presumably initiated anti-inflammatory mechanisms of the 3D spheroids. Notwithstanding that the IL-6 and IL-8 production of 2D cells was mostly not influenced by the endotoxins used, only the higher LTA dose was capable to evoke an IL-8 surge. Flagellin, PMA and poly I:C exerted proinflammatory action in certain concentrations in both 2D and 3D cultures, reflected by the increased cellular IL-6 release. Based on these data, LTA, flagellin, PMA and poly I:C can be considered as potent candidates to induce inflammation in chicken primary hepatic cell cultures, while LPS failed to trigger proinflammatory cytokine production, suggesting the relatively high tolerance of avian liver cells to certain bacterial endotoxins. These results substantiate that the established 3D co-cultures seemed to be proper tools for testing potential proinflammatory molecules; however, the remarkable differences between 2D and 3D models should be addressed and further studied.
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Melzer, Catharina, Juliane von der Ohe, Hannah Otterbein, Hendrik Ungefroren, and Ralf Hass. "Changes in uPA, PAI-1, and TGF-β Production during Breast Cancer Cell Interaction with Human Mesenchymal Stroma/Stem-Like Cells (MSC)." International Journal of Molecular Sciences 20, no. 11 (May 28, 2019): 2630. http://dx.doi.org/10.3390/ijms20112630.
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The interactions of cancer cells with neighboring non-malignant cells in the microenvironment play an important role for progressive neoplastic development and metastasis. Long-term direct co-culture of human MDA-MB-231cherry breast cancer cells with benign human mesenchymal stroma/stem-like cells (MSC) MSC544GFP stably expressing mCherry and eGFP fluorescence proteins, respectively, was associated with the formation of three-dimensional (3D) tumor spheroids in vitro. The quantification of the breast tumor marker urokinase plasminogen activator (uPA) in mono-cultured MDA-MB-231 cells revealed an approximately 14-fold enhanced expression when compared to five different normal human MSC mono-cultures. Moreover, uPA levels in 3D tumor spheroids remained elevated 9.4-fold above the average of five different human MSC cultures. In contrast, the expression of the corresponding plasminogen activator inhibitor type-1 (PAI-1) declined by 2.6-fold in the breast cancer cells and was even further reduced by 3.2-fold in the MDA-MB-231cherry/MSC544GFP 3D co-culture spheroids when compared to the various MSC populations. The supportive data were obtained for the production of TGF-β1, which is an important growth factor in the regulation of tumor growth and metastasis formation. Whereas, TGF-β1 release in MDA-MB-231cherry/MSC544GFP co-cultures was elevated by 1.56-fold as compared to MSC544 mono-cultures after 24 h; this ratio further increased to 2.19-fold after 72 h. Quantitative PCR analyses in MSC544 and MDA-MB-231 cells revealed that MSC, rather than the breast cancer cells, are responsible for TGF-β1 synthesis and that TGF-β1 contributes to its own synthesis in these cells. These findings suggested potential synergistic effects in the expression/secretion of uPA, PAI-1, and TGF-β during the co-culture of breast cancer cells with MSC.
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Konoplina, K. M., E. N. Kosobokova, and V. S. Kosorukov. "Current approaches to assessing the biological activity of immunocytokines <i>in vitro</i>." Russian Journal of Biotherapy 21, no. 3 (October 30, 2022): 10–22. http://dx.doi.org/10.17650/1726-9784-2022-21-3-10-22.
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The use of cytokines as anticancer drugs is limited due to their toxicity. It is possible to reduce toxicity and to increase the therapeutic index by using cytokines in the form of conjugates with antibodies – immunocytokines. The results of preclinical studies have shown increased efficacy and reduced toxicity of various immunocytokines compared to the original, unconjugated cytokines. The potential of immunocytokines as anticancer agents is currently being studied in clinical trials. The therapeutic efficacy of immunocytokines depends on their physicochemical parameters, which determine the in vivo biodistribution, and biological activity as a result of the mechanisms of the antibody action and cytokine sites incorporated in the design. There is a need for methods that allow to assess the biological activity of both individual sites and the entire immunocytokine molecule when characterizing immunocytokines at an early stage of research.This review considers the existing approaches for assessing the biological activity of immunocytokines in vitro in the course of preclinical studies, such as monolayer cultures, reporter cell lines, co-cultures, three-dimensional (3D) tumor models. Monolayer cultures are sufficient to confirm the mechanism of action of separate sites of immunocytokines used in the design, and the “gold standard” test systems for determining the specific biological activity of the cytokine and the effector functions of the antibody site remain in demand. Commercial reporter cell lines remain an alternative option for assessing the biological activity of cytokine and antibody sites at the level of activation of signaling pathways. Co-cultures of tumor and effector cells make it possible to evaluate the cytotoxic and immunomodulatory effects of antibody and cytokine sites without using 3D cultivation methods. The use of 3D tumor models makes it possible to replace several tests for the biological activity of separate sites of immunocytokines conducted on monolayer cultures and co-cultures with one comprehensive study, however, such models require significant time and material costs.
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Pal, Amarnath, Jennifer C. Ashworth, Pamela Collier, Catherine Probert, Sal Jones, Eduardo Pernaut Leza, Marian L. Meakin, et al. "A 3D Heterotypic Breast Cancer Model Demonstrates a Role for Mesenchymal Stem Cells in Driving a Proliferative and Invasive Phenotype." Cancers 12, no. 8 (August 14, 2020): 2290. http://dx.doi.org/10.3390/cancers12082290.
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Previous indirect 2D co-culture studies have demonstrated that mesenchymal stem cells (MSCs) promote breast cancer (BC) progression through secretion of paracrine factors including growth factors, cytokines and chemokines. In order to investigate this aspect of the tumour microenvironment in a more relevant 3D co-culture model, spheroids incorporating breast cancer cells (BCCs), both cell lines and primary BCCs expanded as patient-derived xenografts, and MSCs were established. MSCs in co-cultures were shown to enhance proliferation of estrogen receptor (ER)/progesterone receptor (PR)-positive BCCs. In addition, co-culture resulted in downregulation of E-cadherin in parallel with upregulation of the epithelial-mesenchymal transition (EMT)-relation transcription factor, SNAIL. Cytoplasmic relocalization of ski-related novel protein N (SnON), a negative regulator of transforming growth factor-beta (TGF-β) signalling, and of β-catenin, involved in a number of pathways including Wnt signalling, was also observed in BCCs in co-cultures in contrast to monocultures. In addition, the β-catenin inhibitor, 3-[[(4-methylphenyl)sulfonyl]amino]-benzoic acid methyl ester (MSAB), mediated reduced growth and invasion in the co-cultures. This study highlights the potential role for SnON as a biomarker for BC invasiveness, and the importance of interactions between TGF-β and Wnt signalling, involving SnON. Such pathways may contribute towards identifying possible targets for therapeutic intervention in BC patients.
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Jarrahy, Reza, Weibiao Huang, George H. Rudkin, Jane M. Lee, Kenji Ishida, Micah D. Berry, Modar Sukkarieh, Benjamin M. Wu, Dean T. Yamaguchi, and Timothy A. Miller. "Osteogenic differentiation is inhibited and angiogenic expression is enhanced in MC3T3-E1 cells cultured on three-dimensional scaffolds." American Journal of Physiology-Cell Physiology 289, no. 2 (August 2005): C408—C414. http://dx.doi.org/10.1152/ajpcell.00196.2004.
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Osteogenic differentiation of osteoprogenitor cells in three-dimensional (3D) in vitro culture remains poorly understood. Using quantitative real-time RT-PCR techniques, we examined mRNA expression of alkaline phosphatase, osteocalcin, and vascular endothelial growth factor (VEGF) in murine preosteoblastic MC3T3-E1 cells cultured for 48 h and 14 days on conventional two-dimensional (2D) poly(l-lactide-co-glycolide) (PLGA) films and 3D PLGA scaffolds. Differences in VEGF secretion and function between 2D and 3D culture systems were examined using Western blots and an in vitro Matrigel-based angiogenesis assay. Expression of both alkaline phosphatase and osteocalcin in cells cultured on 3D scaffolds was significantly downregulated relative to 2D controls in 48 h and 14 day cultures. In contrast, elevated levels of VEGF expression in 3D culture were noted at every time point in short- and long-term culture. VEGF protein secretion in 3D cultures was triple the amount of secretion observed in 2D controls. Conditioned medium from 3D cultures induced an enhanced level of angiogenic activity, as evidenced by increases in branch points observed in in vitro angiogenesis assays. These results collectively indicate that MC3T3-E1 cells commit to osteogenic differentiation at a slower rate when cultured on 3D PLGA scaffolds and that VEGF is preferentially expressed by these cells when they are cultured in three dimensions.
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Jones, Dustin P., William Hanna, Gwendolyn M. Cramer, and Jonathan P. Celli. "In situ measurement of ECM rheology and microheterogeneity in embedded and overlaid 3D pancreatic tumor stroma co-cultures via passive particle tracking." Journal of Innovative Optical Health Sciences 10, no. 06 (November 2017): 1742003. http://dx.doi.org/10.1142/s1793545817420032.
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Tumor growth is regulated by a diverse set of extracellular influences, including paracrine crosstalk with stromal partners, and biophysical interactions with surrounding cells and tissues.Studies elucidating the role of physical force and the mechanical properties of the extracellular matrix (ECM) itself as regulators of tumor growth and invasion have been greatly catalyzed by the use of in vitro three-dimensional (3D) tumor models. These systems provide the ability to systematically isolate, manipulate, and evaluate impact of stromal components and extracellular mechanics in a platform that is both conducive to imaging and biologically relevant. However, recognizing that mechanoregulatory crosstalk is bi-directional and fully utilizing these models requires complementary methods for in situ measurements of the local mechanical environment. Here, in 3D tumor/fibroblast co-culture models of pancreatic cancer, a disease characterized by its prominent stromal involvement, we evaluate the use of particle-tracking microrheology to probe dynamic mechanical changes. Using videos of fluorescently labeled polystyrene microspheres embedded in collagen I ECM, we measure spatiotemporal changes in the Brownian motion of probes to report local ECM shear modulus and microheterogeneity. This approach reveals stiffening of collagen in fibroblast co-cultures relative to cultures with cancer cells only, which exhibit degraded ECM with heterogeneous microstructure. We further show that these effects are dependent on culture geometry with contrasting behavior for embedded and overlay cultures. In addition to potential application to screening stroma-targeted therapeutics, this work also provides insight into how the composition and plating geometry impact the mechanical properties of 3D cell cultures that are increasingly widely used in cancer biology.
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Caicedo-Carvajal, Carlos, Qing Liu, Andre Goy, Andrew L. Pecora, Anthony R. Mato, Tatyana Feldman, and K. Stephen Suh. "A Novel 3D Co-Culture System for Isolation and Amplification of Primary Liquid Cancer Cells." Blood 116, no. 21 (November 19, 2010): 426. http://dx.doi.org/10.1182/blood.v116.21.426.426.
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Abstract Abstract 426 One of the important challenges in screening anti-cancer drugs is the lack of available “primary cultures systems” that is easy to use to screen new compounds or their combinations. The low yield of primary cancer cell cultures is mainly due to suboptimum environment in vitro and inefficient 2-dimensional cell culture conditions. To create an optimum in vitro environment, lymphoma cell lines were grown in 3-dimension model by using a scaffold and the stromal cells derived from neonatal foreskin was used as the feeder component. This 3-dimensional (3D) stromal co-culture generates an in-vitro model that may mimic the conditions/microenvironment of blood cancer cells interacting with stromal compartments. A specific 3D tissue culture scaffold 3D Insert-PS™ (300 μ m in fiber diameter and 400μ m in pore size) significantly enhances the cell proliferation and maintenance of liquid cancer cells in comparison to 2D stromal co-culture control. The combination of the neonatal stroma cells, a novel 3D scaffold, the constant gyration and a frequent nutrient stimulation allows the lymphoma cells to proliferate 10-fold faster than the cells grown in 2D under the same condition. Starting from the 2nd day of 3D cell culture, these lymphoma cells grew to form layers of aggregated clusters and caused disappearance of single cells morphology and phenotype that is typical of cells growing in suspension. The cell aggregates are continuously produced from the 3D scaffold, subsequently dislodge from the scaffold and then remain viable at the bottom of the dish below the scaffold. When the cell clusters are harvested and cultured in 3D condition, the contamination of fibroblasts is over 1,000 fold less than the cell clusters that are generated from 2D environment. In addition, the clusters of cancer cells generated from 3D co-culture using 3D scaffolds contained the fibroblasts contamination that is less than 0.00001% of the total cell count, suggesting that this novel 3D environment can be implicated for the isolation of primary lymphoma/cancer cells from patient's blood or tissue specimen. To investigate this feasibility, <1% lymphoma cells were premixed with 100 fold excess of neonatal stroma cells, and the mixture was grown using our 3D scaffolds. In 7 days, the 3D culture system was able to amplify lymphoma cells over 100 fold or over 10,000 % of the starting cell number. This preliminary data indicate that this 3D scaffold and co-culturing environment can be customized to amplify primary cancer cells from blood or tumor tissues and subsequently used for personalized drug screening procedures. Disclosures: Goy: Allos Therapeutics, Inc.: Consultancy, Honoraria.
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Tan, Justin J. Y., Duc-Viet Nguyen, John E. Common, Chunyong Wu, Paul C. L. Ho, and Lifeng Kang. "Investigating PEGDA and GelMA Microgel Models for Sustained 3D Heterotypic Dermal Papilla and Keratinocyte Co-Cultures." International Journal of Molecular Sciences 22, no. 4 (February 21, 2021): 2143. http://dx.doi.org/10.3390/ijms22042143.
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Hair follicle morphogenesis is heavily dependent on reciprocal, sequential, and epithelial-mesenchymal interaction (EMI) between epidermal stem cells and the specialized cells of the underlying mesenchyme, which aggregate to form the dermal condensate (DC) and will later become the dermal papilla (DP). Similar models were developed with a co-culture of keratinocytes and DP cells. Previous studies have demonstrated that co-culture with keratinocytes maintains the in vivo characteristics of the DP. However, it is often challenging to develop three-dimensional (3D) DP and keratinocyte co-culture models for long term in vitro studies, due to the poor intercellular adherence between keratinocytes. Keratinocytes exhibit exfoliative behavior, and the integrity of the DP and keratinocyte co-cultured spheroids cannot be maintained over prolonged culture. Short durations of culture are unable to sufficiently allow the differentiation and re-programming of the keratinocytes into hair follicular fate by the DP. In this study, we explored a microgel array approach fabricated with two different hydrogel systems. Using poly (ethylene glycol) diacrylate (PEGDA) and gelatin methacrylate (GelMA), we compare their effects on maintaining the integrity of the cultures and their expression of important genes responsible for hair follicle morphogenesis, namely Wnt10A, Wnt10B, and Shh, over prolonged duration. We discovered that low attachment surfaces such as PEGDA result in the exfoliation of keratinocytes and were not suitable for long-term culture. GelMA, on the hand, was able to sustain the integrity of co-cultures and showed higher expression of the morphogens overtime.
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Foglietta, Federica, Loredana Serpe, and Roberto Canaparo. "The Effective Combination between 3D Cancer Models and Stimuli-Responsive Nanoscale Drug Delivery Systems." Cells 10, no. 12 (November 25, 2021): 3295. http://dx.doi.org/10.3390/cells10123295.
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Stimuli-responsive drug-delivery systems (DDSs) have emerged as a potential tool for applications in healthcare, mainly in the treatment of cancer where versatile nanocarriers are co-triggered by endogenous and exogenous stimuli. Two-dimensional (2D) cell cultures are the most important in vitro model used to evaluate the anticancer activity of these stimuli-responsive DDSs due to their easy manipulation and versatility. However, some limitations suggest that these in vitro models poorly predict the outcome of in vivo studies. One of the main drawbacks of 2D cell cultures is their inadequate representation of the 3D environment’s physiological complexity, which sees cells interact with each other and the extracellular matrix (ECM) according to their specific cellular organization. In this regard, 3D cancer models are a promising approach that can overcome the main shortcomings of 2D cancer cell cultures, as these in vitro models possess many peculiarities by which they mimic in vivo tumors, including physiologically relevant cell–cell and cell–ECM interactions. This is, in our opinion, even more relevant when a stimuli-responsive DDS is being investigated. In this review, we therefore report and discuss endogenous and exogenous stimuli-responsive DDSs whose effectiveness has been tested using 3D cancer cell cultures.
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Rijal, Girdhari, Chandra Bathula, and Weimin Li. "Application of Synthetic Polymeric Scaffolds in Breast Cancer 3D Tissue Cultures and Animal Tumor Models." International Journal of Biomaterials 2017 (2017): 1–9. http://dx.doi.org/10.1155/2017/8074890.
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Preparation of three-dimensional (3D) porous scaffolds from synthetic polymers is a challenge to most laboratories conducting biomedical research. Here, we present a handy and cost-effective method to fabricate polymeric hydrogel and porous scaffolds using poly(lactic-co-glycolic) acid (PLGA) or polycaprolactone (PCL). Breast cancer cells grown on 3D polymeric scaffolds exhibited distinct survival, morphology, and proliferation compared to those on 2D polymeric surfaces. Mammary epithelial cells cultured on PLGA- or PCL-coated slides expressed extracellular matrix (ECM) proteins and their receptors. Estrogen receptor- (ER-) positive T47D breast cancer cells are less sensitive to 4-hydroxytamoxifen (4-HT) treatment when cultured on the 3D porous scaffolds than in 2D cultures. Finally, cancer cell-laden polymeric scaffolds support consistent tumor formation in animals and biomarker expression as seen in human native tumors. Our data suggest that the porous synthetic polymer scaffolds satisfy the basic requirements for 3D tissue cultures both in vitro and in vivo. The scaffolding technology has appealing potentials to be applied in anticancer drug screening for a better control of the progression of human cancers.
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Monferrer, Ezequiel, Sabina Sanegre, Susana Martín-Vañó, Andrea García-Lizarribar, Rebeca Burgos-Panadero, Amparo López-Carrasco, Samuel Navarro, Josep Samitier, and Rosa Noguera. "Digital Image Analysis Applied to Tumor Cell Proliferation, Aggressiveness, and Migration-Related Protein Synthesis in Neuroblastoma 3D Models." International Journal of Molecular Sciences 21, no. 22 (November 17, 2020): 8676. http://dx.doi.org/10.3390/ijms21228676.
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Patient-derived cancer 3D models are a promising tool that will revolutionize personalized cancer therapy but that require previous knowledge of optimal cell growth conditions and the most advantageous parameters to evaluate biomimetic relevance and monitor therapy efficacy. This study aims to establish general guidelines on 3D model characterization phenomena, focusing on neuroblastoma. We generated gelatin-based scaffolds with different stiffness and performed SK-N-BE(2) and SH-SY5Y aggressive neuroblastoma cell cultures, also performing co-cultures with mouse stromal Schwann cell line (SW10). Model characterization by digital image analysis at different time points revealed that cell proliferation, vitronectin production, and migration-related gene expression depend on growing conditions and are specific to the tumor cell line. Morphometric data show that 3D in vitro models can help generate optimal patient-derived cancer models, by creating, identifying, and choosing patterns of clinically relevant artificial microenvironments to predict patient tumor cell behavior and therapeutic responses.
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Dhimolea, Eugen, and Constantine S. Mitsiades. "Effect of nonmalignant accessory cells from the metastatic microenvironment on breast cancer cell resistance to antiestrogens." Journal of Clinical Oncology 31, no. 15_suppl (May 20, 2013): 11039. http://dx.doi.org/10.1200/jco.2013.31.15_suppl.11039.
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11039 Background: To address the role of the local metastatic microenvironment in the antiestrogen (AE) resistance exhibited by disseminated breast cancer (BrCa), we assembled heterotypic in vitro three-dimensional (3D) tissue cultures comprised of estrogen receptor-positive (ER+) BrCa cells and non-malignant accessory cells from organs frequently targeted by metastatic disease, to model the composition and architecture of metastatic lesions. Methods: MCF7 cells expressing luciferase, cultured alone or with accessory cells, were exposed to dose-ranges of hydroxytamoxifen (4-OHT), fulvestrant or raloxifene. MCF7 cells were also injected s.c. in nude mice either alone or with HS-5 bone marrow stromal cells (BMSCs). Tumor spheroid viability or xenograft growth were measured by bioluminescence. Results: MCF7 response to AEs in 3D conditions (collagen type I and/or Matrigel) was marked by acinar differentiation of tumor spheroids that resembles normal breast epithelium. Co-culture of MCF7 and immortalized accessory cells from the bone (BMSCs; HOBIT and hFOB osteoblasts), brain (SVGp12 astrocytes), liver (THLE3 hepatocytes), and lung (MRC9 fibroblasts) in 3D (but not 2D) conditions, conferred resistance to 4-OHT, raloxifene, and fulvestrant at clinically relevant doses. Heterotypic xenografts (MCF7 + BMSCs) had reduced response to tamoxifen compared with monotypic xenografts (MCF7 alone). BMSCs induced in MCF7 cells downregulation of TGFβ2; upregulation of a transcriptional signature enriched for genes associated with high-grade tumors, including genes involved in interferon response, signaling through EGFR superfamily members, NFkB and other antiapoptotic pathways; and elevated c-Myc protein levels. Exogenous TGFβ2 or neutralizing antibodies against INFγ partially re-sensitized MCF7 cells to 4-OHT in co-cultures. Conclusions: Our results indicate that accessory cells representing the microenvironment of metastatic sites confer AE resistance to BrCa cells in 3D tissue cultures and xenograft models. The mechanisms of stroma-induced AE resistance may reveal new therapeutic targets for refractory BrCa patients with metastatic disease.
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Alsaykhan, Hamad, and Jennifer Z. Paxton. "Investigating materials and orientation parameters for the creation of a 3D musculoskeletal interface co-culture model." Regenerative Biomaterials 7, no. 4 (May 1, 2020): 413–25. http://dx.doi.org/10.1093/rb/rbaa018.
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Abstract Musculoskeletal tissue interfaces are a common site of injury in the young, active populations. In particular, the interface between the musculoskeletal tissues of tendon and bone is often injured and to date, no single treatment has been able to restore the form and function of damaged tissue at the bone–tendon interface. Tissue engineering and regeneration hold great promise for the manufacture of bespoke in vitro models or implants to be used to advance repair and so this study investigated the material, orientation and culture choices for manufacturing a reproducible 3D model of a musculoskeletal interface between tendon and bone cell populations. Such models are essential for future studies focussing on the regeneration of musculoskeletal interfaces in vitro. Cell-encapsulated fibrin hydrogels, arranged in a horizontal orientation though a simple moulding procedure, were shown to best support cellular growth and migration of cells to form an in vitro tendon–bone interface. This study highlights the importance of acknowledging the material and technical challenges in establishing co-cultures and suggests a reproducible methodology to form 3D co-cultures between tendon and bone, or other musculoskeletal cell types, in vitro.
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Haselager, Marco, Eduard Perelaer, Arnon P. Kater, and Eric Eldering. "Development of a Novel Lymph Node-Based 3D Culture System Promoting Chronic Lymphocytic Leukemia Proliferation and Survival." Blood 136, Supplement 1 (November 5, 2020): 47–48. http://dx.doi.org/10.1182/blood-2020-141962.
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INTRODUCTION. Primary chronic lymphocytic leukemia (CLL) cells, despite originating from a proliferative disease, rapidly undergo apoptosis in vitro in absence of microenvironmental survival signals1. Although co-culture with stromal cells or the addition of soluble factors can increase and extend CLL survival, no system permits the long-term expansion of CLL cells in vitro2. The difficulties of mimicking a physiologic microenvironment supporting CLL cells hinder in vitro studies of proliferation, drug screens and prevent propagation of rare subclones. For other cancers, various types of 3D cultures have been introduced utilizing scaffolds, gels, spheroid cultures and fluidic systems, representing a more accurate representation of the in vivo microenvironment3. Unlike solid tumors, secondary lymphoid tissues where CLL cells proliferate in vivo, do not derive from a single stem cell progenitor. Developing an appropriate 3D in vitro culture system for CLL is of obvious importance and may contribute pathophysiological relevance to study long-term CLL proliferation and more accurate drug screening4,5. Within the field of CLL, attempts have focused on bone marrow stroma, but it may be biologically and clinically more relevant to investigate the lymph node niche as this is the critical site of CLL proliferation6. METHODS. Primary CLL cells were cultured in various 3D systems including hydrogels, hanging drop cultures and ultra-low attachment plates (ULA) plates in parallel to an optimal 2D system, consisting of the culture of primary CLL cells on a monolayer of CD40L-presenting fibroblasts (3T40) or 3T3 negative control fibroblasts. CLL cells were either cultured as PBMCs alone, with or without T cells, or co-cultured with 3T40 or primary lymph node fibroblasts. CLL cells were either stimulated directly with IL-2, IL-15, IL-21 and CpG and/or indirectly via a T cell stimulation of anti-CD3/CD28. RESULTS. After testing and comparing multiple systems for the in vitro culture of CLL cells, we optimized a novel CLL culture system utilizing ULA plates creating spheroids of PBMCs isolated from peripheral blood. Without the addition of soluble factors or stroma, primary CLL cells in the ULA 3D model could be maintained in culture for 6 weeks as opposed to 1 week in the 2D system. Aside from significantly promoting CLL survival, cultures could be expanded approximately 3-4-fold over a course of 6 weeks using the ULA 3D model. 3D cultures showed a more consistent and significantly increased CLL proliferation compared to 2D cultures, independent of IGHV mutation status, increasing the average proliferation index of 2.87 to 3.90 (n=10). Additionally, co-culture with LN-derived stromal cells further increased CLL proliferation, reaching a maximum of 8 generations (n=6) (Figure 1). Lastly, when PBMCs were stimulated with IL-2, IL-15, IL-21 and CpG, spheroids developed proliferation center-like structures after 4 weeks of culture. CONCLUSIONS. We established a lymph node-based 3D in vitro culture system for CLL leading to increased CLL proliferation and survival compared to 2D systems. The set-up allows long-term expansion of CLL cells in vitro, as well as formation of proliferation center-like structures. We are currently optimizing drug resistance studies, expansion of specific CLL subclones and performing competition experiments. References: 1. Hamilton et al., Mimicking the tumour microenvironment: three different co-culture systems induce a similar phenotype but distinct proliferative signals in primary chronic lymphocytic leukaemia cells, 2012. 2. Asslaber et al., Mimicking the microenvironment in chronic lymphocytic leukaemia - where does the journey go?, 2013. 3. Gurski et al., 3D Matrices for Anti-Cancer Drug Testing and Development, 2010. 4. Nunes et al., 3D tumor spheroids as in vitro models to mimic in vivo human solid tumors resistance to therapeutic drugs, 2019. 5. Aljitwai et al., A novel three-dimensional stromal-based model for in vitro chemotherapy sensitivity testing of leukemia cells, 2014. 6. Van Gent et al., In vivo dynamics of stable chronic lymphocytic leukemia inversely correlates with somatic hypermutation levels and suggest no major leukemic turnover in bone marrow, 2008. Disclosures Kater: Genentech: Research Funding; Abbvie: Research Funding; Roche: Research Funding; Janssen: Research Funding; Celgene: Research Funding. Eldering:Celgene: Research Funding; Janssen: Research Funding; Genentech: Research Funding.
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Laternser, Sandra, Hansjoerg Keller, Olivier Leupin, Martin Rausch, Ursula Graf-Hausner, and Markus Rimann. "A Novel Microplate 3D Bioprinting Platform for the Engineering of Muscle and Tendon Tissues." SLAS TECHNOLOGY: Translating Life Sciences Innovation 23, no. 6 (June 12, 2018): 599–613. http://dx.doi.org/10.1177/2472630318776594.
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Two-dimensional (2D) cell cultures do not reflect the in vivo situation, and thus it is important to develop predictive three-dimensional (3D) in vitro models with enhanced reliability and robustness for drug screening applications. Treatments against muscle-related diseases are becoming more prominent due to the growth of the aging population worldwide. In this study, we describe a novel drug screening platform with automated production of 3D musculoskeletal-tendon-like tissues. With 3D bioprinting, alternating layers of photo-polymerized gelatin-methacryloyl-based bioink and cell suspension tissue models were produced in a dumbbell shape onto novel postholder cell culture inserts in 24-well plates. Monocultures of human primary skeletal muscle cells and rat tenocytes were printed around and between the posts. The cells showed high viability in culture and good tissue differentiation, based on marker gene and protein expressions. Different printing patterns of bioink and cells were explored and calcium signaling with Fluo4-loaded cells while electrically stimulated was shown. Finally, controlled co-printing of tenocytes and myoblasts around and between the posts, respectively, was demonstrated followed by co-culture and co-differentiation. This screening platform combining 3D bioprinting with a novel microplate represents a promising tool to address musculoskeletal diseases.
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Ferreira Branquinho, Maryana Stephany, Maysa Braga Barros Silva, Gabriela Ansanelo Castilho, Jacqueline Cavalcante, Silvia Berlanga de Moraes Barros, Renan Orsati Clara, Silvya Stuchi Maria‐Engler, and Ana Campa. "Kynurenine inhibits melanogenesis in human melanocyte‐keratinocyte co‐cultures and in a reconstructed 3D skin model." Experimental Dermatology 31, no. 3 (November 6, 2021): 427–32. http://dx.doi.org/10.1111/exd.14486.
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Hwa, Albert J., Rebecca C. Fry, Anand Sivaraman, Peter T. So, Leona D. Samson, Donna B. Stolz, and Linda G. Griffith. "Rat liver sinusoidal endothelial cells survive without exogenous VEGF in 3D perfused co‐cultures with hepatocytes." FASEB Journal 21, no. 10 (April 10, 2007): 2564–79. http://dx.doi.org/10.1096/fj.06-7473com.
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Linde, Nina, Claudia M. Gutschalk, Claudia Hoffmann, Dilan Yilmaz, and Margareta M. Mueller. "Integrating Macrophages into Organotypic Co-Cultures: A 3D In Vitro Model to Study Tumor-Associated Macrophages." PLoS ONE 7, no. 7 (July 6, 2012): e40058. http://dx.doi.org/10.1371/journal.pone.0040058.
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Schneider, Jaana, Marianne Pultar, and Wolfgang Holnthoner. "Ex vivo engineering of blood and lymphatic microvascular networks." Vascular Biology 1, no. 1 (May 3, 2019): H17—H22. http://dx.doi.org/10.1530/vb-19-0012.
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Upon implantation, engineered tissues rely on the supply with oxygen and nutrients as well as the drainage of interstitial fluid. This prerequisite still represents one of the current challenges in the engineering and regeneration of tissues. Recently, different vascularization strategies have been developed. Besides technical approaches like 3D printing or laser processing and de-/recelluarization of natural scaffolds, mainly co-cultures of endothelial cells (ECs) with supporting cell types are being used. This mini-review provides a brief overview of different co-culture systems for the engineering of blood and lymphatic microvascular networks.
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Kordas, Antonis, Phanee Manganas, Alexandros Selimis, Georgios D. Barmparis, Maria Farsari, and Anthi Ranella. "Development of an Oriented Co-Culture System Using 3D Scaffolds Fabricated via Non-Linear Lithography." Materials 15, no. 12 (June 20, 2022): 4349. http://dx.doi.org/10.3390/ma15124349.
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Damage in the Peripheral Nervous System (PNS) is related to numerous neurodegenerative diseases and has consequently drawn the attention of Tissue Engineering (TE), which is considered a promising alternative to already established methods such as surgery and autografts. TE focuses on the design, optimization, and use of scaffolds in vitro and in vivo. In this work, the authors used a novel scaffold geometry fabricated via Multiphoton Lithography (MPL), a commonly used fabrication method, for the mono- and co-cultures of glial Schwann (SW10) and neuronal Neuro-2a (N2a) cells. Both cell types have already been used for the study of various neurodegenerative diseases. However, their focus has been on only one of the cell types at a time, with studies regarding their co-culture only recently documented. Here, the suitability of the fabricated scaffolds has been explored and the effects of topography on SW10 and N2a behavior have been investigated. Our findings demonstrate that scaffold co-culture systems favor the presence of neurites compared to mono-cultures at 21 days (31.4 ± 5.5% and 15.4 ± 5.4%, respectively), while there is also a significant decrease in long neurites in the mono-culture over time (45.3 ± 15.9% at 7 days versus 15.4 ± 5.4% at 21 days). It has been shown that the scaffolds can successfully manipulate cell growth, elongation, and morphology, and these results can form a basis for the development of an experimental model for the study of PNS-related diseases and understanding of key cell functions such as myelination.
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Musi, Clara Alice, Luca Colnaghi, Arianna Giani, Erica Cecilia Priori, Giacomo Marchini, Matteo Tironi, Claudio Conci, et al. "Effect of 3D Synthetic Microscaffold Nichoid on the Morphology of Cultured Hippocampal Neurons and Astrocytes." Cells 11, no. 13 (June 23, 2022): 2008. http://dx.doi.org/10.3390/cells11132008.
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The human brain is the most complex organ in biology. This complexity is due to the number and the intricate connections of brain cells and has so far limited the development of in vitro models for basic and applied brain research. We decided to create a new, reliable, and cost-effective in vitro system based on the Nichoid, a 3D microscaffold microfabricated by two-photon laser polymerization technology. We investigated whether these 3D microscaffold devices can create an environment allowing the manipulation, monitoring, and functional assessment of a mixed population of brain cells in vitro. With this aim, we set up a new model of hippocampal neurons and astrocytes co-cultured in the Nichoid microscaffold to generate brain micro-tissues of 30 μm thickness. After 21 days in culture, we morphologically characterized the 3D spatial organization of the hippocampal astrocytes and neurons within the microscaffold, and we compared our observations to those made using the classical 2D co-culture system. We found that the co-cultured cells colonized the entire volume of the 3D devices. Using confocal microscopy, we observed that within this period the different cell types had become well-differentiated. This was further elaborated with the use of drebrin, PSD-95, and synaptophysin antibodies that labeled the majority of neurons, both in the 2D as well as in the 3D co-cultures. Using scanning electron microscopy, we found that neurons in the 3D co-culture displayed a significantly larger amount of dendritic protrusions compared to neurons in the 2D co-culture. This latter observation indicates that neurons growing in a 3D environment may be more prone to form connections than those co-cultured in a 2D condition. Our results show that the Nichoid can be used as a 3D device to investigate the structure and morphology of neurons and astrocytes in vitro. In the future, this model can be used as a tool to study brain cell interactions in the discovery of important mechanisms governing neuronal plasticity and to determine the factors that form the basis of different human brain diseases. This system may potentially be further used for drug screening in the context of various brain diseases.
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Andreatta, Francesco, Giulia Beccaceci, Nicolò Fortuna, Martina Celotti, Dario De Felice, Marco Lorenzoni, Veronica Foletto, Sacha Genovesi, Josep Rubert, and Alessandro Alaimo. "The Organoid Era Permits the Development of New Applications to Study Glioblastoma." Cancers 12, no. 11 (November 9, 2020): 3303. http://dx.doi.org/10.3390/cancers12113303.
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Glioblastoma (GB) is the most frequent and aggressive type of glioma. The lack of reliable GB models, together with its considerable clinical heterogeneity, has impaired a comprehensive investigation of the mechanisms that lead to tumorigenesis, cancer progression, and response to treatments. Recently, 3D cultures have opened the possibility to overcome these challenges and cerebral organoids are emerging as a leading-edge tool in GB research. The opportunity to easily engineer brain organoids via gene editing and to perform co-cultures with patient-derived tumor spheroids has enabled the analysis of cancer development in a context that better mimics brain tissue architecture. Moreover, the establishment of biobanks from GB patient-derived organoids represents a crucial starting point to improve precision medicine therapies. This review exemplifies relevant aspects of 3D models of glioblastoma, with a specific focus on organoids and their involvement in basic and translational research.
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Pustchi, Sadaf E., Naze G. Avci, Yasemin M. Akay, and Metin Akay. "Astrocytes Decreased the Sensitivity of Glioblastoma Cells to Temozolomide and Bay 11-7082." International Journal of Molecular Sciences 21, no. 19 (September 28, 2020): 7154. http://dx.doi.org/10.3390/ijms21197154.
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Glioblastoma multiforme (GBM) is the most common malignant type of astrocytic tumors. GBM patients have a poor prognosis with a median survival of approximately 15 months despite the “Stupp” Regimen and high tumor recurrence due to the tumor resistance to chemotherapy. In this study, we co-cultured GBM cells with human astrocytes in three-dimensional (3D) poly(ethylene glycol) dimethyl acrylate (PEGDA) microwells to mimic the tumor microenvironment. We treated 3D co- and mono-cultured cells with Temozolomide (TMZ) and the nuclear factor-κB (NF-κB) inhibitor Bay 11-7082 and investigated the combined effect of the drugs. We assessed the expressions of glial fibrillary acidic protein (GFAP) and vimentin that play a role in the tumor malignancy and activation of the astrocytes as well as Notch-1 and survivin that play a role in GBM malignancy after the drug treatment to understand how astrocytes induced GBM drug response. Our results showed that in the co-culture, astrocytes increased GBM survival and resistance after combined drug treatment compared to mono-cultures. These data restated the importance of 3D cell culture to mimic the tumor microenvironment for drug screening.
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Su, Chengxun, Yon Jin Chuah, Hong Boon Ong, Hui Min Tay, Rinkoo Dalan, and Han Wei Hou. "A Facile and Scalable Hydrogel Patterning Method for Microfluidic 3D Cell Culture and Spheroid-in-Gel Culture Array." Biosensors 11, no. 12 (December 10, 2021): 509. http://dx.doi.org/10.3390/bios11120509.
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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.
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Muzzi, L., D. Di Lisa, P. Arnaldi, D. Aprile, L. Pastorino, S. Martinoia, and M. Frega. "Rapid generation of functional engineered 3D human neuronal assemblies: network dynamics evaluated by micro-electrodes arrays." Journal of Neural Engineering 18, no. 6 (December 1, 2021): 066030. http://dx.doi.org/10.1088/1741-2552/ac3e02.
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Abstract Objective. In this work we adapted a protocol for the fast generation of human neurons to build 3D neuronal networks with controlled structure and cell composition suitable for systematic electrophysiological investigations. Approach. We used biocompatible chitosan microbeads as scaffold to build 3D networks and to ensure nutrients-medium exchange from the core of the structure to the external environment. We used excitatory neurons derived from human-induced pluripotent stem cells (hiPSCs) co-cultured with astrocytes. By adapting the well-established NgN2 differentiation protocol, we obtained 3D engineered networks with good control over cell density, volume and cell composition. We coupled the 3D neuronal networks to 60-channel micro electrode arrays (MEAs) to monitor and characterize their electrophysiological development. In parallel, we generated two-dimensional neuronal networks cultured on chitosan to compare the results of the two models. Main results. We sustained samples until 60 d in vitro (DIV) and 3D cultures were healthy and functional. From the structural point of view, the hiPSC derived neurons were able to adhere to chitosan microbeads and to form a stable 3D assembly thanks to the connections among cells. From a functional point of view, neuronal networks showed spontaneous activity after a couple of weeks. Significance. We presented a particular method to generate 3D engineered cultures for the first time with human-derived neurons coupled to MEAs, overcoming some of the limitations related to 2D and 3D neuronal networks and thus increasing the therapeutic target potential of these models for biomedical applications.
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Cho, Sun Wook, Young A. Kim, Hyun Jin Sun, Ye An Kim, Byung-Chul Oh, Ka Hee Yi, Do Joon Park, and Young Joo Park. "CXCL16 signaling mediated macrophage effects on tumor invasion of papillary thyroid carcinoma." Endocrine-Related Cancer 23, no. 2 (November 11, 2015): 113–24. http://dx.doi.org/10.1530/erc-15-0196.
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Macrophages in tumor microenvironment have pivotal roles in tumor growth, metastasis, and angiogenesis. We investigated the interacting mechanism of macrophage actions in human papillary thyroid cancer (PTC). Co-cultures of macrophage/PTC significantly increased the cancer cell migration potentials, compared with the PTC culture alone. Treatment of conditioned medium (CM) of macrophage/PTC co-cultures enhanced cell invasions in 3D invasion assay. Cytokine array analysis demonstrated that CM of macrophage/PTC co-cultures contained a high level of CXCL16, while it was not found in CM of PTC culture alone. Treatment with CXCL16 enhanced the cell migration potentials in PTC cells, and blocking CXCL16 signaling using anti-CXCL16 antibody or metalloproteinase inhibitor (TAPI2) attenuated macrophage-mediated enhancement of PTC cell migration potentials. In PTC cells, CXCL16 treatment or co-cultures with macrophages increased Akt phosphorylation, and these macrophage-dependent increases of Akt phosphorylation was inhibited by anti-CXCL16 antibody. Moreover, Akt inhibitor attenuated macrophage-mediated increases of PTC cell migration potential. In macrophages, treatment of macrophage/PTC co-cultured CMs up-regulated CD163, Il10, and CD206, which were attenuated by anti-CXCL16 antibody treatment. Finally, CXCR6 and CXCL16 expressions were evaluated by immunohistochemical staining with a thyroid tissue microarray including 136 PTC. CXCR6 expressions showed positive correlation with the density of CD163+ macrophages and associated with lymph node metastasis. In conclusion, CXCL16 signaling partly mediated macrophage actions on PTC tumor cell invasion and also changed the macrophage phenotypes into M2-macrophages in PTC tumor microenvironment. These data suggested that CXCL16 signaling, a bidirectional player in macrophage-associated tumor microenvironment, might be a potential therapeutic target of human PTC.
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Krieger, Jessica, Byung-Wook Park, Christopher R. Lambert, and Christopher Malcuit. "3D skeletal muscle fascicle engineering is improved with TGF-β1 treatment of myogenic cells and their co-culture with myofibroblasts." PeerJ 6 (July 11, 2018): e4939. http://dx.doi.org/10.7717/peerj.4939.
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Background Skeletal muscle wound healing is dependent on complex interactions between fibroblasts, myofibroblasts, myogenic cells, and cytokines, such as TGF-β1. This study sought to clarify the impact of TGF-β1 signaling on skeletal muscle cells and discern between the individual contributions of fibroblasts and myofibroblasts to myogenesis when in co-culture with myogenic cells. 3D tissue-engineered models were compared to equivalent 2D culture conditions to assess the efficacy of each culture model to predictively recapitulate the in vivo muscle environment. Methods TGF-β1 treatment and mono-/co-cultures containing human dermal fibroblasts or myofibroblasts and C2C12 mouse myoblasts were assessed in 2D and 3D environments. Three culture systems were compared: cell monolayers grown on 2D dishes and 3D tissues prepared via a self-assembly method or collagen 1-based hydrogel biofabrication. qPCR identified gene expression changes during fibroblast to myofibroblast and myoblast differentiation between culture conditions. Changes to cell phenotype and tissue morphology were characterized via immunostaining for myosin heavy chain, procollagen, and α-smooth muscle actin. Tissue elastic moduli were measured with parallel plate compression and atomic force microscopy systems, and a slack test was employed to quantify differences in tissue architecture and integrity. Results TGF-β1 treatment improved myogenesis in 3D mono- and co-cultures containing muscle cells, but not in 2D. The 3D TGF-β1-treated co-culture containing myoblasts and myofibroblasts expressed the highest levels of myogenin and collagen 1, demonstrating a greater capacity to drive myogenesis than fibroblasts or TGF-β1-treatment in monocultures containing only myoblasts. These constructs possessed the greatest tissue stability, integrity, and muscle fiber organization, as demonstrated by their rapid and sustained shortening velocity during slack tests, and the highest Young’s modulus of 6.55 kPA, approximate half the stiffness of in situ muscle. Both self-assembled and hydrogel-based tissues yielded the most multinucleated, elongated, and aligned muscle fiber histology. In contrast, the equivalent 2D co-culture model treated with TGF-β1 completely lacked myotube formation through suppression of myogenin gene expression. Discussion These results show skeletal muscle regeneration can be promoted by treating myogenic cells with TGF-β1, and myofibroblasts are superior enhancers of myogenesis than fibroblasts. Critically, both TGF-β1 treatment and co-culturing skeletal muscle cells with myofibroblasts can serve as myogenesis accelerators across multiple tissue engineering platforms. Equivalent 2D culture systems cannot replicate these affects, however, highlighting a need to continually improve in vitro models for skeletal muscle development, discovery of therapeutics for muscle regeneration, and research and development of in vitro meat products.
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Krausz, Eberhard, Ronald de Hoogt, Emmanuel Gustin, Frans Cornelissen, Thierry Grand-Perret, Lut Janssen, Nele Vloemans, et al. "Translation of a Tumor Microenvironment Mimicking 3D Tumor Growth Co-culture Assay Platform to High-Content Screening." Journal of Biomolecular Screening 18, no. 1 (August 24, 2012): 54–66. http://dx.doi.org/10.1177/1087057112456874.
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For drug discovery, cell-based assays are becoming increasingly complex to mimic more realistically the nature of biological processes and their diversifications in diseases. Multicellular co-cultures embedded in a three-dimensional (3D) matrix have been explored in oncology to more closely approximate the physiology of the human tumor microenvironment. High-content analysis is the ideal technology to characterize these complex biological systems, although running such complex assays at higher throughput is a major endeavor. Here, we report on adapting a 3D tumor co-culture growth assay to automated microscopy, and we compare various imaging platforms (confocal vs. nonconfocal) with correlating automated image analysis solutions to identify optimal conditions and settings for future larger scaled screening campaigns. The optimized protocol has been validated in repeated runs where established anticancer drugs have been evaluated for performance in this innovative assay.
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Zhang, Ning, Vincent Milleret, Greta Thompson-Steckel, Ning-Ping Huang, János Vörös, Benjamin R. Simona, and Martin Ehrbar. "Soft Hydrogels Featuring In-Depth Surface Density Gradients for the Simple Establishment of 3D Tissue Models for Screening Applications." SLAS DISCOVERY: Advancing the Science of Drug Discovery 22, no. 5 (March 9, 2017): 635–44. http://dx.doi.org/10.1177/2472555217693191.
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Three-dimensional (3D) cell culture models are gaining increasing interest for use in drug development pipelines due to their closer resemblance to human tissues. Hydrogels are the first-choice class of materials to recreate in vitro the 3D extra-cellular matrix (ECM) environment, important in studying cell-ECM interactions and 3D cellular organization and leading to physiologically relevant in vitro tissue models. Here we propose a novel hydrogel platform consisting of a 96-well plate containing pre-cast synthetic PEG-based hydrogels for the simple establishment of 3D (co-)culture systems without the need for the standard encapsulation method. The in-depth density gradient at the surface of the hydrogel promotes the infiltration of cells deposited on top of it. The ability to decouple hydrogel production and cell seeding is intended to simplify the use of hydrogel-based platforms and thus increase their accessibility. Using this platform, we established 3D cultures relevant for studying stem cell differentiation, angiogenesis, and neural and cancer models.