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1
Liu, Qingxi, Zijiang Zhang, Yupeng Liu, Zhanfeng Cui, Tongcun Zhang, Zhaohui Li e Wenjian Ma. "Cancer cells growing on perfused 3D collagen model produced higher reactive oxygen species level and were more resistant to cisplatin compared to the 2D model". Journal of Applied Biomaterials & Functional Materials 16, n. 3 (2 aprile 2018): 144–50. http://dx.doi.org/10.1177/2280800018764763.
Testo completoAbstract (sommario):
Introduction: Three-dimensional (3D) collagen scaffold models, due to their ability to mimic the tissue and organ structure in vivo, have received increasing interest in drug discovery and toxicity evaluation. Methods: In this study, we developed a perfused 3D model and studied cellular response to cytotoxic drugs in comparison with traditional 2D cell cultures as evaluated by cancer drug cisplatin. Results: Cancer cells grown in perfused 3D environments showed increased levels of reactive oxygen species (ROS) production compared to the 2D culture. As determined by growth analysis, cells in the 3D culture, after forming a spheroid, were more resistant to the cancer drug cisplatin compared to that of the 2D cell culture. In addition, 3D culturing cells showed elevated level of ROS, indicating a physiological change or the formation of a microenvironment that resembles tumor cells in vivo. Conclusions: These data revealed that cellular response to drugs for cells growing in 3D environments are dramatically different from that of 2D cultured cells. Thus, the perfused 3D collagen scaffold model we report here might be a potentially very useful tool for drug analysis.
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Chae, Dong-Sik, Sang Joon An, Seongho Han e Sung-Whan Kim. "Synergistic Therapeutic Potential of Dual 3D Mesenchymal Stem Cell Therapy in an Ischemic Hind Limb Mouse Model". International Journal of Molecular Sciences 24, n. 19 (27 settembre 2023): 14620. http://dx.doi.org/10.3390/ijms241914620.
Testo completoAbstract (sommario):
Three-dimensional (3D) culture systems have been widely used to promote the viability and metabolic activity of mesenchymal stem cells (MSCs). The aim of this study was to explore the synergistic benefits of using dual 3D MSC culture systems to promote vascular regeneration and enhance therapeutic potential. We used various experimental assays, including dual 3D cultures of human adipose MSCs (hASCs), quantitative reverse transcription polymerase chain reaction (qRT-PCR), in vitro cell migration, Matrigel tube network formation, Matrigel plug assay, therapeutic assays using an ischemic hind limb mouse model, and immunohistochemical analysis. Our qRT-PCR results revealed that fibroblast growth factor 2 (FGF-2), granulocyte chemotactic protein-2 (GCP-2), and vascular endothelial growth factor-A (VEGF-A) were highly upregulated in conventional 3D-cultured hASCs (ASC-3D) than in two-dimensional (2D)-cultured hASCs. Hepatocyte growth factor (HGF), insulin-like growth factor-1 (IGF-1), and stromal-cell-derived factor-1 (SDF-1) showed higher expression levels in cytokine-cocktail-based, 3D-cultured hASCs (ASC-3Dc). A conditioned medium (CM) mixture of dual 3D ASCs (D-3D; ASC-3D + ASC-3Dc) resulted in higher migration and Matrigel tube formation than the CM of single 3D ASCs (S-3D; ASC-3D). Matrigel plugs containing D-3D contained more red blood cells than those containing S-3D. D-3D transplantation into ischemic mouse hind limbs prevented limb loss and augmented blood perfusion when compared to S-3D transplantation. Transplanted D-3D also revealed a high capillary density and angiogenic cytokine levels and transdifferentiated into endothelial-like cells in the hind limb muscle. These findings highlight the benefits of using the dual 3D culture system to optimize stem-cell-based therapeutic strategies, thereby advancing the therapeutic strategy for ischemic vascular disease and tissue regeneration.
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Silva, Emmanuel João Nogueira Leal, Nancy Kudsi de Carvalho, Carina Taboada Ronconi, Gustavo De-Deus, Mario Luis Zuolo e Alexandre Augusto Zaia. "Cytotoxicity Profile of Endodontic Sealers Provided by 3D Cell Culture Experimental Model". Brazilian Dental Journal 27, n. 6 (dicembre 2016): 652–56. http://dx.doi.org/10.1590/0103-6440201600792.
Testo completoAbstract (sommario):
Abstract The aim of the present study was to evaluate the cytotoxic effects of five endodontic sealers (AH Plus, Endomethasone N, EndoSequence BC, MTA Fillapex and Pulp Canal Sealer EWT) using a three-dimensional (3D) cell culture model. A conventional bi-dimensional (2D) cell culture model was used as reference technique for comparison. Balb/c 3T3 fibroblasts were cultured in conventional bi-dimensional cell culture and in rat-tail collagen type I three-dimensional cell culture models. Then, both cell cultures were incubated with elutes of freshly mixed endodontic sealers for 24 h. Cell viability was measured by the methyl-thiazol-diphenyltetrazolium assay (MTT). Data were statistically analyzed using ANOVA and the Tukey test at a significance level of p<0.05. All tested sealers exhibited cytotoxic effects; however, cytotoxic effect was culture model- and sealer-dependent. Sealers showed higher cytotoxicity in 2D than in 3D cell culture model (p<0.05). In both conditions, EndoSequence BC showed the lowest cytotoxicity (p<0.05). MTA Fillapex was much more cytotoxic than the other tested endodontic sealers (p<0.05), with the exception of AH Plus in the 2D cell culture model (p>0.05). Endomethasone N and Pulp Canal Sealer EWT showed lower cytotoxic effects than AH Plus in 2D cell culture model (p<0.05); however no statistical differences was observed among these sealers in 3D cell culture model. It may be concluded that cytotoxicity was higher in 2D cell culture compared to 3D cell culture. EndoSequence BC sealer exhibited the highest cytocompatibility and MTA Fillapex the lowest cytocompatibility.
4
Kreß, Sebastian, Roland Schaller-Ammann, Jürgen Feiel, Joachim Wegener, Joachim Priedl, Wolf Dietrich, Cornelia Kasper e Dominik Egger. "Innovative Platform for the Advanced Online Monitoring of Three-Dimensional Cells and Tissue Cultures". Cells 11, n. 3 (25 gennaio 2022): 412. http://dx.doi.org/10.3390/cells11030412.
Testo completoAbstract (sommario):
The use of 3D cell cultures has gained increasing importance in medical and pharmaceutical research. However, the analysis of the culture medium is hardly representative for the culture conditions within a 3D model which hinders the standardization of 3D cultures and translation of results. Therefore, we developed a modular monitoring platform combining a perfusion bioreactor with an integrated minimally invasive sampling system and implemented sensors that enables the online monitoring of culture parameters and medium compounds within 3D cultures. As a proof-of-concept, primary cells as well as cell lines were cultured on a collagen or gelatin methacryloyl (GelMA) hydrogel matrix, while monitoring relevant culture parameters and analytes. Comparing the interstitial fluid of the 3D models versus the corresponding culture medium, we found considerable differences in the concentrations of several analytes. These results clearly demonstrate that analyses of the culture medium only are not relevant for the development of standardized 3D culture processes. The presented bioreactor with an integrated sampling and sensor platform opens new horizons for the development, optimization, and standardization of 3D cultures. Furthermore, this technology holds the potential to reduce animal studies and improve the transferability of pharmaceutical in vitro studies by gaining more relevant results, bridging the gap towards clinical translation.
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Rosendahl, Jennifer, Andreas Svanström, Mattias Berglin, Sarunas Petronis, Yalda Bogestål, Patrik Stenlund, Simon Standoft et al. "3D Printed Nanocellulose Scaffolds as a Cancer Cell Culture Model System". Bioengineering 8, n. 7 (10 luglio 2021): 97. http://dx.doi.org/10.3390/bioengineering8070097.
Testo completoAbstract (sommario):
Current conventional cancer drug screening models based on two-dimensional (2D) cell culture have several flaws and there is a large need of more in vivo mimicking preclinical drug screening platforms. The microenvironment is crucial for the cells to adapt relevant in vivo characteristics and here we introduce a new cell culture system based on three-dimensional (3D) printed scaffolds using cellulose nanofibrils (CNF) pre-treated with 2,2,6,6-tetramethylpyperidine-1-oxyl (TEMPO) as the structural material component. Breast cancer cell lines, MCF7 and MDA-MB-231, were cultured in 3D TEMPO-CNF scaffolds and were shown by scanning electron microscopy (SEM) and histochemistry to grow in multiple layers as a heterogenous cell population with different morphologies, contrasting 2D cultured mono-layered cells with a morphologically homogenous cell population. Gene expression analysis demonstrated that 3D TEMPO-CNF scaffolds induced elevation of the stemness marker CD44 and the migration markers VIM and SNAI1 in MCF7 cells relative to 2D control. T47D cells confirmed the increased level of the stemness marker CD44 and migration marker VIM which was further supported by increased capacity of holoclone formation for 3D cultured cells. Therefore, TEMPO-CNF was shown to represent a promising material for 3D cell culture model systems for cancer cell applications such as drug screening.
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Bauer, Magdalena, Magdalena Metzger, Marvin Corea, Barbara Schädl, Johannes Grillari e Peter Dungel. "Novel 3D-Printed Cell Culture Inserts for Air–Liquid Interface Cell Culture". Life 12, n. 8 (10 agosto 2022): 1216. http://dx.doi.org/10.3390/life12081216.
Testo completoAbstract (sommario):
In skin research, widely used in vitro 2D monolayer models do not sufficiently mimic physiological properties. To replace, reduce, and refine animal experimentation in the spirit of ‘3Rs’, new approaches such as 3D skin equivalents (SE) are needed to close the in vitro/in vivo gap. Cell culture inserts to culture SE are commercially available, however, these inserts are expensive and of limited versatility regarding experimental settings. This study aimed to design novel cell culture inserts fabricated on commercially available 3D printers for the generation of full-thickness SE. A computer-aided design model was realized by extrusion-based 3D printing of polylactic acid filaments (PLA). Improvements in the design of the inserts for easier and more efficient handling were confirmed in cell culture experiments. Cytotoxic effects of the final product were excluded by testing the inserts in accordance with ISO-norm procedures. The final versions of the inserts were tested to generate skin-like 3D scaffolds cultured at an air–liquid interface. Stratification of the epidermal component was demonstrated by histological analyses. In conclusion, here we demonstrate a fast and cost-effective method for 3D-printed inserts suitable for the generation of 3D cell cultures. The system can be set-up with common 3D printers and allows high flexibility for generating customer-tailored cell culture plastics.
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Takahashi, Yuki, Yumi Nomura, Yuma Yokokawa, Shiro Kitano, Satoshi Nagayama, Eiji Shinozaki, Ryohei Katayama e Naoya Fujita. "Abstract 4565: Drug screening by layered 3D co-cultured tumor model including vascularized stromal tissue". Cancer Research 83, n. 7_Supplement (4 aprile 2023): 4565. http://dx.doi.org/10.1158/1538-7445.am2023-4565.
Testo completoAbstract (sommario):
Abstract Introduction: In vivo, tumor microenvironments consist of not only cancer cells but also extracellular matrix and stromal tissues, such as fibroblasts, blood vessels, and so on. The interactions between cancer cells and stromal tissue have been reported to affect the behavior of cancer cells. So that ex vivo model recapturing the tumor microenvironment is needed to evaluate the efficacy of drugs under the condition mimicking the patient tumor tissue. Here, we developed the unique tissue engineering technique, which easily enables the construction of cell - stacked three dimensional (3D) tissue, and co-culture of 3D stromal tissues and patient-derived cancer cells (PDCs). We investigated drug sensitivity in conventional 2D culture, our 3D co-cultured model and in vivo tumor. Methods: Fibroblasts and vascular endothelial cells were suspended in a buffer solution containing heparin and collagen to support cell aggregation. The heparin/collagen-treated cells were seeded in culture-inserts in over-confluent manner, and 3D layered stromal tissue called were constructed. PDCs established from colorectal cancer (CRC) and non-small cell lung cancer (NSCLC) patients in the Cancer Institute Hospital of JFCR were co-cultured with the 3D stromal tissue. The 3D co-cultured model applied to drug screening, and the results were compared with those of 2D culture model. In vivo drug evaluations were performed with the compounds in which marked differences were observed between 2D and 3D models. Results: In our 3D model, drug sensitivities to most of the tested compounds tended to be decreased in comparison with those in 2D culture condition. Interestingly, a part of drugs did not effective in 2D showed marked tumor growth inhibition in our 3D model. The compounds that showed favorable efficacy in 3D rather than 2D in multiple PDCs were accounted for about 5% of tested compounds. At least half of these drugs showed significant tumor growth suppression or tumor regression in vivo. On the contrary, in the case of drug sensitivities were considerably fallen in our 3D model, most of the evaluated compounds represented almost no anti-tumor effect in vivo. Results from gene and protein expression analyses supported that cancer cells co-cultured in our 3D stromal tissue have some similar profiles to in vivo tumor rather than 2D culture condition. Conclusion: Our study proposed the unique 3D co-cultured tumor model. The model may enable more accurate drug screening reflecting the in vivo circumstances. Further studies are needed to confirm the model’s predictability of clinical outcomes. Citation Format: Yuki Takahashi, Yumi Nomura, Yuma Yokokawa, Shiro Kitano, Satoshi Nagayama, Eiji Shinozaki, Ryohei Katayama, Naoya Fujita. Drug screening by layered 3D co-cultured tumor model including vascularized stromal tissue. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 4565.
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Scalise, Mariangela, Fabiola Marino, Luca Salerno, Nunzia Amato, Claudia Quercia, Chiara Siracusa, Andrea Filardo et al. "Adult Multipotent Cardiac Progenitor-Derived Spheroids: A Reproducible Model of In Vitro Cardiomyocyte Commitment and Specification". Cells 12, n. 13 (5 luglio 2023): 1793. http://dx.doi.org/10.3390/cells12131793.
Testo completoAbstract (sommario):
Background: Three-dimensional cell culture systems hold great promise for bridging the gap between in vitro cell-based model systems and small animal models to study tissue biology and disease. Among 3D cell culture systems, stem-cell-derived spheroids have attracted significant interest as a strategy to better mimic in vivo conditions. Cardiac stem cell/progenitor (CSC)-derived spheroids (CSs) provide a relevant platform for cardiac regeneration. Methods: We compared three different cell culture scaffold-free systems, (i) ultra-low attachment plates, (ii) hanging drops (both requiring a 2D/3D switch), and (iii) agarose micro-molds (entirely 3D), for CSC-derived CS formation and their cardiomyocyte commitment in vitro. Results: The switch from a 2D to a 3D culture microenvironment per se guides cell plasticity and myogenic differentiation within CS and is necessary for robust cardiomyocyte differentiation. On the contrary, 2D monolayer CSC cultures show a significant reduced cardiomyocyte differentiation potential compared to 3D CS culture. Forced aggregation into spheroids using hanging drop improves CS myogenic differentiation when compared to ultra-low attachment plates. Performing CS formation and myogenic differentiation exclusively in 3D culture using agarose micro-molds maximizes the cardiomyocyte yield. Conclusions: A 3D culture system instructs CS myogenic differentiation, thus representing a valid model that can be used to study adult cardiac regenerative biology.
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Metelmann, Isabella B., Sebastian Kraemer, Matthias Steinert, Stefan Langer, Peggy Stock e Olga Kurow. "Novel 3D organotypic co-culture model of pleura". PLOS ONE 17, n. 12 (1 dicembre 2022): e0276978. http://dx.doi.org/10.1371/journal.pone.0276978.
Testo completoAbstract (sommario):
Pleural mesothelial cells are the predominant cell type in the pleural cavity, but their role in the pathogenesis of pleural diseases needs to be further elucidated. 3D organotypic models are an encouraging approach for an in vivo understanding of molecular disease development. The aim of the present study was to develop a 3D organotypic model of the pleural mesothelium. Specimens of human pleura parietalis were obtained from patients undergoing surgery at the University Hospital Leipzig, Germany. 3D co-culture model of pleura was established from human pleural mesothelial cells and fibroblasts. The model was compared to human pleura tissue by phase-contrast and light microscopy, immunochemistry and -fluorescence as well as solute permeation test. Histological assessment of the 3D co-culture model displayed the presence of both cell types mimicking the morphology of the human pleura. Vimentin and Cytokeratin, PHD1 showed a similar expression pattern in pleural biopsies and 3D model. Expression of Ki-67 indicates the presence of proliferating cells. Tight junctional marker ZO-1 was found localized at contact zones between mesothelial cells. Each of these markers were expressed in both the 3D co-culture model and human biopsies. Permeability of 3D organotypic co-culture model of pleura was found to be higher for 70 kDa-Dextran and no significant difference was seen in the permeability for small dextran (4 kDa). In summary, the presented 3D organoid of pleura functions as a robust assay for pleural research serving as a precise reproduction of the in vivo morphology and microenvironment.
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Prosser, Amy, Colin Scotchford, George Roberts, David Grant e Virginie Sottile. "Integrated Multi-Assay Culture Model for Stem Cell Chondrogenic Differentiation". International Journal of Molecular Sciences 20, n. 4 (22 febbraio 2019): 951. http://dx.doi.org/10.3390/ijms20040951.
Testo completoAbstract (sommario):
Recent osteochondral repair strategies highlight the promise of mesenchymal progenitors, an accessible stem cell source with osteogenic and chondrogenic potential, used in conjunction with biomaterials for tissue engineering. For this, regenerative medicine approaches require robust models to ensure selected cell populations can generate the desired cell type in a reproducible and measurable manner. Techniques for in vitro chondrogenic differentiation are well-established but largely qualitative, relying on sample staining and imaging. To facilitate the in vitro screening of pro-chondrogenic treatments, a 3D micropellet culture combined with three quantitative GAG assays has been developed, with a fourth parallel assay measuring sample content to enable normalisation. The effect of transforming growth factor beta (TGF-β) used to validate this culture format produced a measurable increase in proteoglycan production in the parallel assays, in both 2D and 3D culture configurations. When compared to traditional micropellets, the monolayer format appeared less able to detect changes in cell differentiation, however in-well 3D cultures displayed a significant differential response. Effects on collagen 2 expression confirmed these observations. Based on these results, a microplate format was optimised for 3D culture, in a high-throughput in-well configuration. This model showed improved sensitivity and confirmed the 3D micropellet in-well quantitative assays as an effective differentiation format compatible with streamlined, high-throughput chondrogenic screens.
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dos Santos, Kelvin Sousa, Lariane Teodoro Oliveira, Marina de Lima Fontes, Ketylin Fernanda Migliato, Ana Marisa Fusco-Almeida, Maria José Soares Mendes Giannini e Andrei Moroz. "Alginate-Based 3D A549 Cell Culture Model to Study Paracoccidioides Infection". Journal of Fungi 9, n. 6 (31 maggio 2023): 634. http://dx.doi.org/10.3390/jof9060634.
Testo completoAbstract (sommario):
A three-dimensional (3D) lung aggregate model based on sodium alginate scaffolds was developed to study the interactions between Paracoccidioides brasiliensis (Pb) and lung epithelial cells. The suitability of the 3D aggregate as an infection model was examined using cell viability (cytotoxicity), metabolic activity, and proliferation assays. Several studies exemplify the similarity between 3D cell cultures and living organisms, which can generate complementary data due to the greater complexity observed in these designed models, compared to 2D cell cultures. A 3D cell culture system of human A549 lung cell line plus sodium alginate was used to create the scaffolds that were infected with Pb18. Our results showed low cytotoxicity, evidence of increased cell density (indicative of cell proliferation), and the maintenance of cell viability for seven days. The confocal analysis revealed viable yeast within the 3D scaffold, as demonstrated in the solid BHI Agar medium cultivation. Moreover, when ECM proteins were added to the alginate scaffolds, the number of retrieved fungi was significantly higher. Our results highlight that this 3D model may be promising for in vitro studies of host–pathogen interactions.
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Kappelmann-Fenzl, Melanie, Sonja K. Schmidt, Stefan Fischer, Rafael Schmid, Lisa Lämmerhirt, Lena Fischer, Stefan Schrüfer et al. "Molecular Changes Induced in Melanoma by Cell Culturing in 3D Alginate Hydrogels". Cancers 13, n. 16 (15 agosto 2021): 4111. http://dx.doi.org/10.3390/cancers13164111.
Testo completoAbstract (sommario):
Alginate hydrogels have been used as a biomaterial for 3D culturing for several years. Here, gene expression patterns in melanoma cells cultivated in 3D alginate are compared to 2D cultures. It is well-known that 2D cell culture is not resembling the complex in vivo situation well. However, the use of very intricate 3D models does not allow performing high-throughput screening and analysis is highly complex. 3D cell culture strategies in hydrogels will better mimic the in vivo situation while they maintain feasibility for large-scale analysis. As alginate is an easy-to-use material and due to its favorable properties, it is commonly applied as a bioink component in the growing field of cell encapsulation and biofabrication. Yet, only a little information about the transcriptome in 3D cultures in hydrogels like alginate is available. In this study, changes in the transcriptome based on RNA-Seq data by cultivating melanoma cells in 3D alginate are analyzed and reveal marked changes compared to cells cultured on usual 2D tissue culture plastic. Deregulated genes represent valuable cues to signaling pathways and molecules affected by the culture method. Using this as a model system for tumor cell plasticity and heterogeneity, EGR1 is determined to play an important role in melanoma progression.
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Takahashi, Yuki, Shiro Kitano, Eiji Shinozaki, Satoshi Nagayama, Ryohei Katayama e Naoya Fujita. "Abstract 6027: Layered 3D co-cultured tumor model including vascularized stromal tissue may reflect drug sensitivities in vivo tumor". Cancer Research 82, n. 12_Supplement (15 giugno 2022): 6027. http://dx.doi.org/10.1158/1538-7445.am2022-6027.
Testo completoAbstract (sommario):
Abstract Background: Multiple 3D culture models have been reported as a superior tumor model than 2D culture. The importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and resistance to treatment. Therefore, ex vivo model recapturing the tumor microenvironment is needed to evaluate the drug efficacy under the condition imitating the patient tumor tissue. Purpose: We developed a unique 3D co-cultured tumor model with stromal tissues containing a microvascular network. Here, we investigated drug sensitivity in conventional 2D culture, our 3D co-cultured model and in vivo tumor. Methods: Drug sensitivity and gene expression on our model were evaluated using patient-derived cancer cells (PDC) established from colorectal cancer (CRC) patients in JFCR. The characteristics were compared with those of conventional 2D cultured cells or patient-derived xenograft (PDX). Results: In our 3D model, the drug sensitivities tended to be decreased in comparison with those of 2D. However, some drugs presented potent efficacy in our 3D model rather than 2D, and such drugs showed significant tumor growth suppression or tumor regression in vivo. Transcriptome profiles of our 3D model showed relatively higher similarity to those of in vivo tumors than those of 2D. Conclusion: Our study proposed the unique 3D co-cultured tumor model which may enable more accurate evaluation of drug sensitivities reflecting the in vivo circumstances. Citation Format: Yuki Takahashi, Shiro Kitano, Eiji Shinozaki, Satoshi Nagayama, Ryohei Katayama, Naoya Fujita. Layered 3D co-cultured tumor model including vascularized stromal tissue may reflect drug sensitivities in vivo tumor [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2022; 2022 Apr 8-13. Philadelphia (PA): AACR; Cancer Res 2022;82(12_Suppl):Abstract nr 6027.
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Ao, Di-Shu, Yun-e. Xu, Xin-Sun, Hui-Fang Cheng, Heng-Mei Li, Xian Yu, Feng-Li Peng et al. "Establishing a three-dimensional culture model of adenovirus using nanoself-assembling peptide KLD-12 hydrogels as scaffolds to evaluate the antiviral effects of IFNα2b". Materials Express 12, n. 3 (1 marzo 2022): 487–97. http://dx.doi.org/10.1166/mex.2022.2164.
Testo completoAbstract (sommario):
Viral infectious diseases seriously threaten human health. At present, the prevention and treatment of viral diseases depends primarily on vaccines and drugs. Commonly used research method include animal models or simple traditional two-dimensional (2D) isolation culture, but 2D cell behaviour is different from the human physiological microenvironment. Therefore, a new model that can simulate the human microenvironment is needed. Here, the nanoself-assembled peptide KLD-12 was used as a culture scaffold for 293T cells in threedimensional (3D) adenovirus culture. As a new 3D virus culture model, it simulates in vivo virus infection, and the model can produce infectious particles. In addition, the antiviral drug sensitivity between the 3D and 2D cultures was significantly different. We established a 3D adenovirus culture model that can be used for adenovirus proliferation and antiviral drug screening, as well as for gene therapy, vaccine research and other research. Additionally, it can partially replace animal models.
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Han, Bo, William Fang, Zhi Yang, Yuntao Wang, Shuqing Zhao, Ba Xuan Hoang e C. Thomas Vangsness. "Enhancement of Chondrogenic Markers by Exosomes Derived from Cultured Human Synovial Fluid-Derived Cells: A Comparative Analysis of 2D and 3D Conditions". Biomedicines 11, n. 12 (25 novembre 2023): 3145. http://dx.doi.org/10.3390/biomedicines11123145.
Testo completoAbstract (sommario):
Objective: The goal of this pilot study was to investigate the effects of exosomes derived from synovial fluid-derived cells (SFDCs) cultured under normoxic conditions in a two-dimensional (2D) monolayer or encapsulated within a three-dimensional (3D) matrix for chondrogenic differentiation in vitro and cartilage defect repair in vivo. Design: Synovial fluid samples were obtained from three patients, and SFDCs were isolated and expanded either in a 2D monolayer culture or seeded within a transglutaminase cross-linked gelatin (Col-Tgel) to create a 3D gel culture. Exosomes derived from each environment were isolated and characterized. Then, their effects on cartilage-cell proliferation and chondrogenic differentiation were assessed using an in vitro organoid model, and their potential for enhancing cartilage repair was evaluated using a rat cartilage defect model. Results: SFDCs obtained from different donors reached a state of senescence after four passages in 2D culture. However, transferring these cells to a 3D culture environment mitigated the senescence and improved cell viability. The 3D-cultured exosomes exhibited enhanced potency in promoting chondrogenic differentiation, as evidenced by the increased expression of chondrogenic genes and greater deposition of cartilage-specific extracellular matrix. Furthermore, the 3D-cultured exosomes demonstrated superior effectiveness in enhancing cartilage repair and exhibited better healing properties compared to exosomes derived from a 2D culture. Conclusions: The optimized 3D culture provided a more favorable environment for the proliferation of human synovial cells and the secretion of exosomes compared to the 2D culture. The 3D-cultured exosomes exhibited greater potential for promoting chondrogenic gene expression in vitro and demonstrated improved healing properties in repairing cartilage defects compared to exosomes derived from the 2D culture.
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Pereira, Vitória Mattos, Priscila Avelino Ferreira Pinto, Lina Castelo Branco Motta, Matheus F. Almeida, André Furugen Cesar de Andrade, Ana Paula Pinoti Pavaneli e Carlos Eduardo Ambrósio. "Initial Characterization of 3D Culture of Yolk Sac Tissue". Animals 13, n. 9 (22 aprile 2023): 1435. http://dx.doi.org/10.3390/ani13091435.
Testo completoAbstract (sommario):
The role of the yolk sac (YS) in miscarriage is not yet clear, largely due to ethical reasons that make in vivo studies difficult to conduct. However, 3D cultures could provide a solution to this problem by enabling cells to be arranged in a way that more closely mimics the structure of the YS as it exists in vivo. In this study, three domestic species (porcine, canine, and bovine) were chosen as models to standardize 3D culture techniques for the YS. Two techniques of 3D culture were chosen: the Matrigel® and Hanging-Drop techniques, and the 2D culture technique was used as a standardized method. The formed structures were initially characterized using scanning electron microscopy (SEM), immunohistochemistry (IHC), and quantitative real-time PCR (RT-qPCR). In general, the 3D culture samples showed better organization of the YS cells compared to 2D cultures. The formed structures from both 3D methods assemble the mesothelial layer of YS tissue. Regarding the IHC assay, all in vitro models were able to express zinc and cholesterol transport markers, although only 3D culture techniques were able to generate structures with different markers pattern, indicating a cell differentiation process when compared to 2D cultures. Regarding mRNA expression, the 3D models had a greater gene expression pattern on the Hemoglobin subunit zeta-like (HBZ) gene related to the YS tissue, although no significant expression was found in Alpha-fetoprotein (AFP), indicating a lack of endodermal differentiation in our 3D model. With the initial technique and characterization established, the next step is to maintain the cultures and characterize the diversity of cell populations, stemness, functions, and genetic stability of each 3D in vitro model.
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Cardona-Mendoza, Andrés, Nelly Stella Roa Molina, Diana Marcela Castillo, Gloria Inés Lafaurie e Diego Fernando Gualtero Escobar. "Human Coronary Artery Endothelial Cell Response to Porphyromonas gingivalis W83 in a Collagen Three-Dimensional Culture Model". Microorganisms 12, n. 2 (24 gennaio 2024): 248. http://dx.doi.org/10.3390/microorganisms12020248.
Testo completoAbstract (sommario):
P. gingivalis has been reported to be an endothelial cell inflammatory response inducer that can lead to endothelial dysfunction processes related to atherosclerosis; however, these studies have been carried out in vitro in cell culture models on two-dimensional (2D) plastic surfaces that do not simulate the natural environment where pathology develops. This work aimed to evaluate the pro-inflammatory response of human coronary artery endothelial cells (HCAECs) to P. gingivalis in a 3D cell culture model compared with a 2D cell culture. HCAECs were cultured for 7 days on type I collagen matrices in both cultures and were stimulated at an MOI of 1 or 100 with live P. gingivalis W83 for 24 h. The expression of the genes COX-2, eNOS, and vWF and the levels of the pro-inflammatory cytokines thromboxane A2 (TXA-2) and prostaglandin I2 (PGI2) were evaluated. P. gingivalis W83 in the 2D cell culture increased IL-8 levels at MOI 100 and decreased MCP-1 levels at both MOI 100 and MOI 1. In contrast, the 3D cell culture induced an increased gene expression of COX-2 at both MOIs and reduced MCP-1 levels at MOI 100, whereas the gene expression of eNOS, vWF, and IL-8 and the levels of TXA2 and PGI2 showed no significant changes. These data suggest that in the collagen 3D culture model, P. gingivalis W83 induces a weak endothelial inflammatory response.
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Sułkowski, Łukasz. "A 3D model and typology of organisational culture". Journal of Intercultural Management 5, n. 2 (1 giugno 2013): 17–30. http://dx.doi.org/10.2478/joim-2013-0008.
Testo completoAbstract (sommario):
Abstract The proposed model of organisational cultures I used in my research is based on three dichotomous dimensions borrowed from G. Hofstede and other researchers. Although Hofstede proposed studying organisational cultures according to other dimensions of values than in the case of cultures of whole societies, there are numerous authors who think his model is more general and so apply it to organisational cultures too. It seems that three out of five dimensions proposed by Hofstede can become a basis for such a multidimensional model and typology. I am also in favour of this approach, as I believe that three of the dimensions included in Hofstede’s model are of a universal character, whether they concern individuals, organisational cultures or social cultures [Sułkowski 2012, pp. 103-118]
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Suominen, Siiri, Tinja Hyypijev, Mari Venäläinen, Alma Yrjänäinen, Hanna Vuorenpää, Mari Lehti-Polojärvi, Mikko Räsänen et al. "Improvements in Maturity and Stability of 3D iPSC-Derived Hepatocyte-like Cell Cultures". Cells 12, n. 19 (27 settembre 2023): 2368. http://dx.doi.org/10.3390/cells12192368.
Testo completoAbstract (sommario):
Induced pluripotent stem cell (iPSC) technology enables differentiation of human hepatocytes or hepatocyte-like cells (iPSC-HLCs). Advances in 3D culturing platforms enable the development of more in vivo-like liver models that recapitulate the complex liver architecture and functionality better than traditional 2D monocultures. Moreover, within the liver, non-parenchymal cells (NPCs) are critically involved in the regulation and maintenance of hepatocyte metabolic function. Thus, models combining 3D culture and co-culturing of various cell types potentially create more functional in vitro liver models than 2D monocultures. Here, we report the establishment of 3D cultures of iPSC-HLCs alone and in co-culture with human umbilical vein endothelial cells (HUVECs) and adipose tissue-derived mesenchymal stem/stromal cells (hASCs). The 3D cultures were performed as spheroids or on microfluidic chips utilizing various biomaterials. Our results show that both 3D spheroid and on-chip culture enhance the expression of mature liver marker genes and proteins compared to 2D. Among the spheroid models, we saw the best functionality in iPSC-HLC monoculture spheroids. On the contrary, in the chip system, the multilineage model outperformed the monoculture chip model. Additionally, the optical projection tomography (OPT) and electrical impedance tomography (EIT) system revealed changes in spheroid size and electrical conductivity during spheroid culture, suggesting changes in cell–cell connections. Altogether, the present study demonstrates that iPSC-HLCs can successfully be cultured in 3D as spheroids and on microfluidic chips, and co-culturing iPSC-HLCs with NPCs enhances their functionality. These 3D in vitro liver systems are promising human-derived platforms usable in various liver-related studies, specifically when using patient-specific iPSCs.
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Zhang, Mei, Philip Boughton, Barbara Rose, C. Soon Lee e Angela M. Hong. "The Use of Porous Scaffold as a Tumor Model". International Journal of Biomaterials 2013 (2013): 1–9. http://dx.doi.org/10.1155/2013/396056.
Testo completoAbstract (sommario):
Background. Human cancer is a three-dimensional (3D) structure consisting of neighboring cells, extracellular matrix, and blood vessels. It is therefore critical to mimic the cancer cells and their surrounding environment duringin vitrostudy. Our aim was to establish a 3D cancer model using a synthetic composite scaffold.Methods. High-density low-volume seeding was used to promote attachment of a non-small-cell lung cancer cell line (NCI-H460) to scaffolds. Growth patterns in 3D culture were compared with those of monolayers. Immunohistochemistry was conducted to compare the expression of Ki67, CD44, and carbonic anhydrase IX.Results. NCI-H460 readily attached to the scaffold without surface pretreatment at a rate of 35% from a load of 1.5 × 106cells. Most cells grew vertically to form clumps along the surface of the scaffold, and cell morphology resembled tissue origin; 2D cultures exhibited characteristics of adherent epithelial cancer cell lines. Expression patterns of Ki67, CD44, and CA IX varied markedly between 3D and monolayer cultures.Conclusions. The behavior of cancer cells in our 3D model is similar to tumor growthin vivo. This model will provide the basis for future study using 3D cancer culture.
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De Vita, Alessandro, Federica Recine, Giacomo Miserocchi, Federica Pieri, Anna Farnedi, Francesco Fabbri, Valentina Fausti et al. "The potential role of extracellular matrix in the activity of trabectedin." Journal of Clinical Oncology 38, n. 15_suppl (20 maggio 2020): e23542-e23542. http://dx.doi.org/10.1200/jco.2020.38.15_suppl.e23542.
Testo completoAbstract (sommario):
e23542 Background: Soft tissue sarcomas (sts) represent a rare group of solid neoplasm of mesenchymal origin with a 1% incidence of all adult cancers. Liposarcoma and leiomyosarcoma (L-sarcoma) are the most common histotypes. The current first-line treatment for advanced/metastatic L-sarcomas is represented by anthracycline based-regimens. Second-line therapy options include trabectedin, eribulin, ifosfamide, gemcitabine and dacarbazine. In particular, the activity of trabectedin, a tetrahydroisoquinoline molecule, as well as its mechanism of action is not completely elucidated. Methods: The study involved six patient affected L-sarcoma. Patient-derived primary cultures were established after patient surgical treatment. Primary cells were cultured in standard monolayer culture and 3D collagen-based scaffolds. In order to gain inside into the mechanism of action of the drugs, genomic-, chemobiogram, proteomic-, cytometer and in silico analysis were performed. Results: The results confirmed the preservation of tumor gene expression in 3D culture model compared to standard monolayer culture. In particular 3D culture system was able to resemble the patient gene expression of TGFb, SLUG, SNAIL, MMP9 compared to 2D. In silico analysis showed an upregulation of COL1A1 gene, the extracellular matrix component of the present 3D model, in sts compared to other tumors. Pharmacological analysis displayed an increased sensitivity to trabectedin in all the 3D L-sarcoma primary culture compared to 2D culture system, while this was not observed with the other drugs. As previous preclinical evidences suggested that trabectedin mechanism of action include the impairing of ECM proteolytic degradation mediated by tumor cells, we think that its high observed sensitivity depend on the presence of collagen in our 3D- model. Conclusions: These results were suggestive of the losing credibility of monolayer standard culture for pharmacology studies and highlight the promising role of 3D patient-derived culture model for the study of sts biology. Moreover these results shed the light on the potential role of ECM in the mechanism of action of trabectedin in L-sarcomas. The work points out the role of this tumor microenvironment component in predicting response to trabectedin and provide the rationale for better stratifying patients which will be candidate for this drug. Further researches are needed to confirm these evidences.
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Sun, Gaoying, Wenwen Liu, Zhaomin Fan, Daogong Zhang, Yuechen Han, Lei Xu, Jieyu Qi et al. "The Three-Dimensional Culture System with Matrigel and Neurotrophic Factors Preserves the Structure and Function of Spiral Ganglion NeuronIn Vitro". Neural Plasticity 2016 (2016): 1–15. http://dx.doi.org/10.1155/2016/4280407.
Testo completoAbstract (sommario):
Whole organ culture of the spiral ganglion region is a resourceful model system facilitating manipulation and analysis of live sprial ganglion neurons (SGNs). Three-dimensional (3D) cultures have been demonstrated to have many biomedical applications, but the effect of 3D culture in maintaining the SGNs structure and function in explant culture remains uninvestigated. In this study, we used the matrigel to encapsulate the spiral ganglion region isolated from neonatal mice. First, we optimized the matrigel concentration for the 3D culture system and found the 3D culture system protected the SGNs against apoptosis, preserved the structure of spiral ganglion region, and promoted the sprouting and outgrowth of SGNs neurites. Next, we found the 3D culture system promoted growth cone growth as evidenced by a higher average number and a longer average length of filopodia and a larger growth cone area. 3D culture system also significantly elevated the synapse density of SGNs. Last, we found that the 3D culture system combined with neurotrophic factors had accumulated effects in promoting the neurites outgrowth compared with 3D culture or NFs treatment only groups. Together, we conclude that the 3D culture system preserves the structure and function of SGN in explant culture.
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Sabetta, Samantha, Davide Vecchiotti, Letizia Clementi, Mauro Di Vito Nolfi, Francesca Zazzeroni e Adriano Angelucci. "Comparative Analysis of Dasatinib Effect between 2D and 3D Tumor Cell Cultures". Pharmaceutics 15, n. 2 (21 gennaio 2023): 372. http://dx.doi.org/10.3390/pharmaceutics15020372.
Testo completoAbstract (sommario):
Three-dimensional cell culture methods are able to confer new predictive relevance to in vitro tumor models. In particular, the 3D multicellular tumor spheroids model is considered to better resemble tumor complexity associated with drug resistance compared to the 2D monolayer model. Recent advances in 3D printing techniques and suitable biomaterials have offered new promises in developing 3D tissue cultures at increased reproducibility and with high-throughput characteristics. In our study, we compared the sensitivity to dasatinib treatment in two different cancer cell lines, prostate cancer cells DU145 and glioblastoma cells U87, cultured in the 3D spheroids model and in the 3D bioprinting model. DU145 and U87 cells were able to proliferate in 3D alginate/gelatin bioprinted structures for two weeks, forming spheroid aggregates. The treatment with dasatinib demonstrated that bioprinted cells were considerably more resistant to drug toxicity than corresponding cells cultured in monolayer, in a way that was comparable to behavior observed in the 3D spheroids model. Recovery and analysis of cells from 3D bioprinted structures led us to hypothesize that dasatinib resistance was dependent on a scarce penetrance of the drug, a phenomenon commonly reported also in spheroids. In conclusion, the 3D bioprinted model utilizing alginate/gelatin hydrogel was demonstrated to be a suitable model in drug screening when spheroid growth is required, offering advantages in feasibility, reproducibility, and scalability compared to the classical 3D spheroids model.
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Mišković Špoljarić, Katarina, Marijana Jukić, Teuta Opačak-Bernardi e Ljubica Glavaš-Obrovac. "3D Cell Technology in Biomedical Research". Collegium antropologicum 44, n. 3 (2020): 171–74. http://dx.doi.org/10.5671/ca.44.3.10.
Testo completoAbstract (sommario):
Traditional two dimensional cell culture has enabled great strides in biomedicine but needs to be improved to be able to keep up with the demands of modern biomedical research. 2D monolayer culture cannot replicate tissue responses and needs to be supplemented with extensive animal research. Growing cells in three dimensional scaffolds provides a more functional model for biomedical research than traditional monolayer culture. Depending on the needs and the complexity of the model there are several ways that 3D models can be initiated. Simple spheroids can be grown in low adherence plates and in hanging drops while larger spheroids and co-cultured ones need to be grown in systems with greater support such as hydro gels. The system that offers the greatest flexibility is the magnetic levitation approach. In the paper we offer a brief resume to various 3D methods and their characteristics to ease the choice of methods for implementing 3D cell culture techniques.
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Strand, Zoe, Finn Schrickel, Sophie Dobiasch, Andreas R. Thomsen, Katja Steiger, Jens Gempt, Bernhard Meyer, Stephanie E. Combs e Daniela Schilling. "Establishment of a 3D Model to Characterize the Radioresponse of Patient-Derived Glioblastoma Cells". Cancers 15, n. 16 (10 agosto 2023): 4051. http://dx.doi.org/10.3390/cancers15164051.
Testo completoAbstract (sommario):
Glioblastoma multiforme (GBM) is the most common malignant primary brain tumor in adults. Despite modern, multimodal therapeutic options of surgery, chemotherapy, tumor-treating fields (TTF), and radiotherapy, the 5-year survival is below 10%. In order to develop new therapies, better preclinical models are needed that mimic the complexity of a tumor. In this work, we established a novel three-dimensional (3D) model for patient-derived GBM cell lines. To analyze the volume and growth pattern of primary GBM cells in 3D culture, a CoSeedisTM culture system was used, and radiation sensitivity in comparison to conventional 2D colony formation assay (CFA) was analyzed. Both culture systems revealed a dose-dependent reduction in survival, but the high variance in colony size and shape prevented reliable evaluation of the 2D cultures. In contrast, the size of 3D spheroids could be measured accurately. Immunostaining of spheroids grown in the 3D culture system showed an increase in the DNA double-strand-break marker γH2AX one hour after irradiation. After 24 h, a decrease in DNA damage was observed, indicating active repair mechanisms. In summary, this new translational 3D model may better reflect the tumor complexity and be useful for analyzing the growth, radiosensitivity, and DNA repair of patient-derived GBM cells.
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Aulthouse, Amy L., Ellen Freeh, Sabrina Newstead e Amy L. Stockert. "Part 1: A Novel Model for Three-Dimensional Culture of 3T3-L1 Preadipocytes Stimulates Spontaneous Cell Differentiation Independent of Chemical Induction Typically Required in Monolayer". Nutrition and Metabolic Insights 12 (gennaio 2019): 117863881984139. http://dx.doi.org/10.1177/1178638819841399.
Testo completoAbstract (sommario):
Differences in monolayer and three-dimensional (3D) culture systems have been recognized for several years. Despite the recognized importance of 3D systems, low cost and convenience of monolayer culture are still readily used for metabolic and nutritional studies. Here, we present part 1 of a 2-part series that will highlight (1) a novel and cost-effective model for culturing 3T3-L1 preadipocytes in 3D agarose as well as (2) an initial study showing the successful use of this 3D model for experimental analysis of these cells treated with cinnamon extract while suspended in agarose. In part 1, we provide a full characterization of the model system for the 3T3-L1 cells that demonstrate the functionality and convenience of this system. Importantly, we note spontaneous differentiation to adipocytes while cultured under these methods, independent of chemical induction. We present a 2.5-week time course with rounded cells forming vacuoles as early as 24 hours and accumulation of lipid detectable by Oil Red O stain at 0.5 weeks. Serum selection, lipid volume determination, and cell size are characterized. We conclusively demonstrate adipogenesis based on a peroxisome proliferator-activated receptor γ (PPARγ) detection using immunohistochemistry (IHC) of sections from these 3D cultures. Methods, materials and recommendations are described as well as proposed benefits to the use of this culture system for 3T3-L1 cells.
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Takahashi, Yuki, Kei Tsukamoto, Rii Morimura, Isana Nada, Yuki Shimizu, Ryohei Katayama, Eiji Shinozaki et al. "A unique ex vivo tumor model: 3D cocultured system with cancer and stromal cells including blood microvessels." Journal of Clinical Oncology 38, n. 4_suppl (1 febbraio 2020): 211. http://dx.doi.org/10.1200/jco.2020.38.4_suppl.211.
Testo completoAbstract (sommario):
211 Background: Importance of interaction between cancer and stromal cells has been widely recognized in tumor progression and tolerance against treatment. Although 2D culture and spheroid consisting only cancer cells still remains the preferred platform for most laboratory preclinical studies while these provide only limited information about tumor microenvironment. In order to mimic the patient tumor tissue, ex vivo model which recaptures the tumor microenvironment is required. Methods: Layered 3D stromal tissues comprising microvascular network were produced by culturing fibroblasts and endothelial cells coated with the extra-cellular matrix (ECM) and natural polysaccharide, namely collagen and heparin. The layered 3D stromal tissues and co-cultured tumor were morphologically evaluated by HE stain, immunohistochemistry and immunofluorescence (IF). Their gene expression and secretome profile were characterized by RNA-sequencing and bio-plex suspension array technologies. Furthermore, drug sensitivity assay were conducted using popular colorectal cancer cell lines, and patient-derived cell lines (PDCs) established in the laboratory of JFCR. Remaining cancer cells post drug treatment were quantified by IF and imaging analysis. Results: The 3D stromal tissues including CD31 positive luminal structure were multi-layered (approximately 20 layers), and the tendency that dense microvascular network was formed nearby cancer cells was observed. In comparison with 2D culture or 3D mono-cultured spheroid model, decreased drug sensitivities were represented in the layered 3D co-cultured model. Omics profiles difference among models suggest that our 3D model has some similarity to in vivo tumor. Conclusions: We developed the layered 3D stromal tissue culture system including blood micro-vessels. Drug sensitivity in the co-cultured tumors may reflect the response of cancer cells in in vivo. Our unique 3D ex vivo model appear to be a valuable tool for drug evaluation, and thus testing approved and/or developing compounds with patient-derived cells would enable better prediction their efficacy.
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Dragoj, Miodrag, Jasmina Stojkovska, Tijana Stanković, Jelena Dinić, Ana Podolski-Renić, Bojana Obradović e Milica Pešić. "Development and Validation of a Long-Term 3D Glioblastoma Cell Culture in Alginate Microfibers as a Novel Bio-Mimicking Model System for Preclinical Drug Testing". Brain Sciences 11, n. 8 (31 luglio 2021): 1025. http://dx.doi.org/10.3390/brainsci11081025.
Testo completoAbstract (sommario):
Background: Various three-dimensional (3D) glioblastoma cell culture models have a limited duration of viability. Our aim was to develop a long-term 3D glioblastoma model, which is necessary for reliable drug response studies. Methods: Human U87 glioblastoma cells were cultured in alginate microfibers for 28 days. Cell growth, viability, morphology, and aggregation in 3D culture were monitored by fluorescent and confocal microscopy upon calcein-AM/propidium iodide (CAM/PI) staining every seven days. The glioblastoma 3D model was validated using temozolomide (TMZ) treatments 3 days in a row with a recovery period. Cell viability by MTT and resistance-related gene expression (MGMT and ABCB1) by qPCR were assessed after 28 days. The same TMZ treatment schedule was applied in 2D U87 cell culture for comparison purposes. Results: Within a long-term 3D model system in alginate fibers, U87 cells remained viable for up to 28 days. On day 7, cells formed visible aggregates oriented to the microfiber periphery. TMZ treatment reduced cell growth but increased drug resistance-related gene expression. The latter effect was more pronounced in 3D compared to 2D cell culture. Conclusion: Herein, we described a long-term glioblastoma 3D model system that could be particularly helpful for drug testing and treatment optimization.
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Kihara, Takanori, Chiya Umezu, Karin Sawada e Yukari Furutani. "Osteogenic cells form mineralized particles, a few μm in size, in a 3D collagen gel culture". PeerJ 7 (23 ottobre 2019): e7889. http://dx.doi.org/10.7717/peerj.7889.
Testo completoAbstract (sommario):
Osteogenic cells form mineralized matrices in vitro, as well as in vivo. The formation and shape of the mineralized matrices are highly regulated by the cells. In vitro formation of mineralized matrices by osteogenic cells can be a model for in vivo osteogenesis. In this study, using a three-dimensional (3D) collagen gel culture system, we developed a new in vitro model for the formation of mineralized particles, a few µm in size, by the osteogenic cells. Human osteosarcoma (HOS) cells formed spherical mineralized matrices (about 12 µm) at approximately 7 days when cultured with β-glycerophosphate (β-GP)-containing culture media on 2D tissue culture plates. Alternately, when they were cultured in a 3D collagen gel containing β-GP, they formed mineralized particles with about 1.7 µm in the gel at approximately 3 days. Calcium precipitation in the gel was evaluated by measuring the gel turbidity. This type of mineralization of HOS cells, which formed mineralized particles inside the gel, was also observed in a peptide-based hydrogel culture. The mineralized particles were completely diminished by inhibiting the activity of Pit-1, phosphate cotransporter, of the HOS cells. When mouse osteoblast-like MC3T3-E1 cells, which form large and flat mineralized matrices in 2D osteogenic conditions at approximately 3 weeks of culture, were cultured in a 3D collagen gel, they also formed mineralized particles in the gel, similar to those in HOS cells, at approximately 18 days. Thus, osteogenic cells cultured in the 3D collagen gel form mineralized particles over a shorter period, and the mineralization could be easily determined by gel turbidity. This 3D gel culture system of osteogenic cells acts as a useful model for cells forming particle-type mineralized matrices, and we assume that the mineralized particles in the 3D hydrogel are calcospherulites, which are derived from matrix vesicles secreted by osteogenic cells.
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Martinez-Armenta, Carlos, Carlos Suarez-Ahedo, Anell Olivos-Meza, María C. Camacho-Rea, Laura E. Martínez-Gómez, Guadalupe Elizabeth Jimenez-Gutierrez, Gabriela A. Martínez-Nava, Luis E. Gomez-Quiroz, Carlos Pineda e Alberto López-Reyes. "The Critical Role of Hypoxia in the Re-Differentiation of Human Articular Chondrocytes". Cells 11, n. 16 (17 agosto 2022): 2553. http://dx.doi.org/10.3390/cells11162553.
Testo completoAbstract (sommario):
The preservation of the chondrogenic phenotype and hypoxia-related physiological microenvironment are major challenges in the 2D culture of primary human chondrocytes. To address this problem, we develop a 3D culture system generating scaffold-free spheroids from human chondrocytes. Our results highlight the chondrogenic potential of cultured human articular chondrocytes in a 3D system combined with hypoxia independently of the cartilage source. After 14 days of culture, we developed spheroids with homogenous diameter and shape from hyaline cartilage donors. Spheroids generated in hypoxia showed a significantly increased glycosaminoglycans synthesis and up-regulated the expression of SOX9, ACAN, COL2A1, COMP, and SNAI1 compared to those obtained under normoxic conditions. Therefore, we conclude that spheroids developed under hypoxic conditions modulate the expression of chondrogenesis-related genes and native tissue features better than 2D cultures. Thus, this scaffold-free 3D culture system represents a novel in vitro model that can be used for cartilage biology research.
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Hardt, Melina, Kurt Zatloukal e Helmut H. Popper. "Abstract 3614: 3D model to study migration and invasion of lung cancer". Cancer Research 83, n. 7_Supplement (4 aprile 2023): 3614. http://dx.doi.org/10.1158/1538-7445.am2023-3614.
Testo completoAbstract (sommario):
Abstract Background: In 2D cell cultures, migration of tumor cells is characterized by epithelial-to-mesenchymal transition (EMT), whereby tumor cells loose epithelial and express mesenchymal markers and very often change into a spindle cell phenotype. In contrast to 2D cultures, in 3D cultures tumor cells do not lose their differentiation. Small cell lung cancer usually moves as single cells or in small clusters, squamous cell and adenocarcinomas preferentially move in large clusters of cells. In tissue specimen, all of these carcinomas migrate in a hybrid EMT, not loosing their cytokeratin and E-cadherin expression. In AC and SCC also polarity and specification do occur, as some tumor cells act as leaders, providing orientation for the followers. However, 3D culture system might better depict real migration and come closer to the in vivo system as 2D cell culture systems. Material and Method: A 3D bioprinter (TissueLabsR) was used to spot an alveolar-mimicking lung-specific matrix on cell culture wells. Microscopic channels out of pluronicsR are created within the matrix. Pluronics is removed by cooling, leaving empty channels within the protein matrix. These channels can be filled with different interleukins, such as IL23, which will direct migration of the tumor cells. Cultured cell lines from an adenocarcinoma (A549) and small cell carcinoma (NCI-H82) are layered on top of the matrix and migration into the matrix is studied using different time points. Results and Discussion: A matrix simulating an alveolar structure was created with MatrigelR. The A549 cells were seeded on the matrix and grown for 2,5, 10, and 14 days. The cells formed cell clusters on the matrix and after one week started to invade the matrix in cell complexes. By immunohistochemistry we could show, that the A549 cells retained their epithelial differentiation. The H82 cells showed a different behavior. They did not form large clusters, but started to invade in small groups. The use of the bioprinter enabled us not only to create a 3D tumor culture system to simulate the in vivo conditions but also to create matrix structures for studying invasion and migration and to stimulate carcinoma cells with migration-activating substances. Citation Format: Melina Hardt, Kurt Zatloukal, Helmut H. Popper. 3D model to study migration and invasion of lung cancer. [abstract]. In: Proceedings of the American Association for Cancer Research Annual Meeting 2023; Part 1 (Regular and Invited Abstracts); 2023 Apr 14-19; Orlando, FL. Philadelphia (PA): AACR; Cancer Res 2023;83(7_Suppl):Abstract nr 3614.
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Zhang, Xuan, Ming-Gen Hu, Ke Pan, Chong-Hui Li e Rong Liu. "3D Spheroid Culture Enhances the Expression of Antifibrotic Factors in Human Adipose-Derived MSCs and Improves Their Therapeutic Effects on Hepatic Fibrosis". Stem Cells International 2016 (2016): 1–8. http://dx.doi.org/10.1155/2016/4626073.
Testo completoAbstract (sommario):
Three-dimensional (3D) cell culture has been reported to increase the therapeutic potentials of mesenchymal stem cells (MSCs). However, the action mechanisms of 3D MSCs vary greatly and are far from being thoroughly investigated. In this study, we aimed to investigate the therapeutic effects of 3D spheroids of human adipose-derived MSCs for hepatic fibrosis. Our results showed that 3D culture enhanced the expression of antifibrotic factors by MSCs, including insulin growth factor 1 (IGF-1), interleukin-6 (IL-6), and hepatocyte growth factor (HGF).In vitrostudies indicated conditioned medium of 3D cultured MSCs protected hepatocytes from cell injury and apoptosis more effectively compared with 2D cultured cells. More importantly, when transplanted into model mice with hepatic fibrosis, 3D spheroids of MSCs were more beneficial in ameliorating hepatic fibrosis and improving liver function than 2D cultured cells. Therefore, the 3D culture strategy improved the therapeutic effects of MSCs and might be promising for treatment of hepatic fibrosis.
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Whyard, Terry, Jingxuan Liu, Frank S. Darras, Wayne C. Waltzer e Victor Romanov. "Organoid model of urothelial cancer: establishment and applications for bladder cancer research". BioTechniques 69, n. 3 (settembre 2020): 193–99. http://dx.doi.org/10.2144/btn-2020-0068.
Testo completoAbstract (sommario):
3D cancer cell models are suitable for drug evaluation because they more precisely mimic tissue architecture than 2D cultures. To study cytotoxicity of anticancer agents, the most sensitive CellTiter-Glo 3D assay is used. However, this is an end point assay, so it is not possible to consider the variance of the starting material amount in the final reading. It is difficult to maintain an even plating density of 3D organoids for cytotoxicity analysis. We present a simple, 3D bladder cancer culture that can be maintained, cryopreserved and used for molecular and drug response studies. We applied a simple modification of the drug response assay for 3D cultures by measuring the background signal with the CellTiter Blue assay before drug application.
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Caicedo-Carvajal, Carlos, Qing Liu, Andre Goy, Andrew L. Pecora, Anthony R. Mato, Tatyana Feldman e K. Stephen Suh. "A Novel 3D Co-Culture System for Isolation and Amplification of Primary Liquid Cancer Cells". Blood 116, n. 21 (19 novembre 2010): 426. http://dx.doi.org/10.1182/blood.v116.21.426.426.
Testo completoAbstract (sommario):
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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Ali, Ahmed S. M., Johanna Berg, Viola Roehrs, Dongwei Wu, Johannes Hackethal, Albert Braeuning, Lisa Woelken, Cornelia Rauh e Jens Kurreck. "Xeno-Free 3D Bioprinted Liver Model for Hepatotoxicity Assessment". International Journal of Molecular Sciences 25, n. 3 (2 febbraio 2024): 1811. http://dx.doi.org/10.3390/ijms25031811.
Testo completoAbstract (sommario):
Three-dimensional (3D) bioprinting is one of the most promising methodologies that are currently in development for the replacement of animal experiments. Bioprinting and most alternative technologies rely on animal-derived materials, which compromises the intent of animal welfare and results in the generation of chimeric systems of limited value. The current study therefore presents the first bioprinted liver model that is entirely void of animal-derived constituents. Initially, HuH-7 cells underwent adaptation to a chemically defined medium (CDM). The adapted cells exhibited high survival rates (85–92%) after cryopreservation in chemically defined freezing media, comparable to those preserved in standard medium (86–92%). Xeno-free bioink for 3D bioprinting yielded liver models with high relative cell viability (97–101%), akin to a Matrigel-based liver model (83–102%) after 15 days of culture. The established xeno-free model was used for toxicity testing of a marine biotoxin, okadaic acid (OA). In 2D culture, OA toxicity was virtually identical for cells cultured under standard conditions and in CDM. In the xeno-free bioprinted liver model, 3-fold higher concentrations of OA than in the respective monolayer culture were needed to induce cytotoxicity. In conclusion, this study describes for the first time the development of a xeno-free 3D bioprinted liver model and its applicability for research purposes.
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Seno, Kotomi, Yasuhisa Munakata, Michiya Sano, Ryouka Kawahara-Miki, Hironori Takahashi, Akihide Ohkuchi, Hisataka Iwata, Takehito Kuwayama e Koumei Shirasuna. "Aggregation of Human Trophoblast Cells into Three-Dimensional Culture System Enhances Anti-Inflammatory Characteristics through Cytoskeleton Regulation". International Journal of Molecular Sciences 19, n. 8 (8 agosto 2018): 2322. http://dx.doi.org/10.3390/ijms19082322.
Testo completoAbstract (sommario):
Background: Three-dimensional (3D) culture changes cell characteristics and function, suggesting that 3D culture provides a more physiologically relevant environment for cells compared with 2D culture. We investigated the differences in cell functions depending on the culture model in human trophoblast cells (Sw.71). Methods: Sw.71 cells were incubated in 2D monolayers or simple 3D spheroids. After incubation, cells were corrected to assess RNA-seq transcriptome or protein expression, and culture medium were corrected to detect cytokines. To clarify the role of actin cytoskeleton, spheroid Sw.71 cells were treated mycalolide B (inhibitor of actin polymerization) in a 3D culture. Results: RNA-seq transcriptome analysis, results revealed that 3D-cultured cells had a different transcriptional profile compared with 2D-cultured cells, especially regarding inflammation-related molecules. Although interleukin-6 (IL-6) mRNA level was higher in 3D-culured cells, its secretion levels were higher in 2D-cultured cells. In addition, the levels of mRNA and protein expression of regnase-1, regulatory RNase of inflammatory cytokine, significantly increased in 3D culture, suggesting post-translational modification of IL-6 mRNA via regnase-1. Treatment with mycalolide B reduced cell-to-cell contact to build 3D formation and increased expression of actin cytoskeleton, resulting in increased IL-6 secretin. Conclusion: Cell dimensionality plays an essential role in governing the spatiotemporal cellular outcomes, including inflammatory cytokine production and its negative regulation associated with regnase-1.
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Aljitawi, Omar S., Dandan Li, Da Zhang, Jonathan Mahnken, Suman Kambhampati, Peter Van Veldhuizen e Rama Garimella. "A 3 Dimensional in Vitro Model to Test HL-60 Cell Line Sensitivity to Chemotherapy". Blood 118, n. 21 (18 novembre 2011): 4882. http://dx.doi.org/10.1182/blood.v118.21.4882.4882.
Testo completoAbstract (sommario):
Abstract Abstract 4882 Introduction: Current in vitro drug testing models are based on 2-dimensional (2D) cell culture systems and therefore do not always predict in vivo responses. This lack of predictability of the 2D assays is believed to be related to the 3-dimensional (3D) microenvironment present in tissues or tumors. This 3D microenvironment, were cell-cell and cell-extracellular matrix (ECM) interactions occur, is fundamental for cell biologic activities. This is especially true for acute myeloid leukemia, were current 2-D cell culture models do not always predict clinical responses. This discrepancy in leukemia cell responses to chemotherapy in vivo, in comparison to in vitro, is at least partly related to leukemia cells interaction with the bone marrow microenvironment and their ability to establish niches. These niches offer partial protection from the effects of cytotoxic chemotherapy, otherwise termed cell adhesion-mediated drug resistance. In these experiments, we investigate the apoptotic effects of cytotoxic chemotherapy on HL-60 cell line cultured in a designed 3D AML cell culture model. In this 3D microenvironment, HL-60 cells were co-cultured with ex vivo expanded bone marrow mesenchyaml stem cells in a 3D synthetic scaffold. Aim: To examine the apoptotic effect of cytotoxic chemotherapy on HL-60 co-cultured with human bone marrow mesenchymal stem cells (huBM-MSCs) in 3D conditions. Methods: After several passages, expanded huBM-MSCs were seeded into PGA/PLLA 90/10 copolymer discs, 5-mm in diameter and 2-mm in thickness and allowed to attach to scaffold fibers and to expand over 2 weeks. Then, HL-60 were added and allowed to grow in the 3D culture system for another 10 days. HL-60 cells in 3D culture system were then exposed to doxorubicin given in two concentrations (25 and 50 μM) and incubated for 24 hours. HL-60 were then retrieved applying a combination of mechanical forces and using cell dissociation solution. FITC Annexin V Apoptosis Detection Kit was used to determine apoptosis. Apoptosis was confirmed by TUNEL assay. Proliferation of HL-60 cells in the 3D scaffold was assessed using Ki-67 stain of scaffold's cryosections. All tests were done in triplicates, and untreated HL-60 served as controls for treatment. Comparison was made with HL-60 cells alone and with HL-60 cells growing on a hu-BM-MSC monolayer. SAS version 9.2 (SAS Institute, Inc., 2002–2008) was used for statistical analysis Results: Virtually, all HL-60 cells treated with 25 or 50 μM underwent late apoptosis. Around.03% of HL-60 cells survived 25 μM concentration, none, however, survived 50 μM concentration. In 2D, most of HL-60 cells underwent necrosis, and to lesser extent late apoptosis. In sharp contrast, 17.8% of HL-60 cells survived 25μM concentration, nevertheless, only.27% of HL-60 cells treated with 50 μM concentration survived. The differences in apoptosis patterns between the three groups was statistically significant (P<.0001). Conclusion: compared to traditional cell culture conditions, the designed 3D culture conditions protected a higher percentage of HL-60 cells from undergoing apoptosis and necrosis. Disclosures: No relevant conflicts of interest to declare.
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Takir, Gizem Gulevin, Bilge Debelec-Butuner e Kemal Sami Korkmaz. "3D Cell Culture Model for Prostate Cancer Cells to Mimic Inflammatory Microenvironment". Proceedings 2, n. 25 (6 dicembre 2018): 1555. http://dx.doi.org/10.3390/proceedings2251555.
Testo completoAbstract (sommario):
The studies on the relationship between inflammation and cancer progression have been mostly carried out with monolayer cell cultures in vitro, which can be insufficient to mimic tumor tissue. Here, we established a three-dimensional (3D) cell culture model of inflammatory microenvironment for prostate cancer cells to better evaluate the role of inflammation in prostate carcinogenesis. Formation of the cell spheroids has been achieved for LNCaP, Du145, LNCaP-104r2 prostate cancer cell lines but not for RWPE1 normal prostate epithelial cell and PC3 by using 3D Petri Dish®. We also showed that cells in inflammatory conditioned media might have a different response based on the culturing method. Overall, we are suggesting that 3D cell culture model can be a useful tool to study molecular alterations on proliferation and migration/invasion of tumor cells related to inflammation.
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Chudakova, D. A., E. Yu Skorova e I. V. Reshetov. "Practical Applications of 3D Cell Culture Biotechnologies for Translational Oncology and Personalized Therapy". Biotekhnologiya 36, n. 3 (2020): 3–15. http://dx.doi.org/10.21519/0234-2758-2020-36-3-3-15.
Testo completoAbstract (sommario):
The creation of in vitro three-dimensional cellular model systems (in vitro 3D cultures) is a fast-growing leading-edge segment of the biotechnological industry. We have examined in this work the key 80 articles published after 2008, and focused on applications of in vitro 3D culture in translational oncology. We described a broad range of 3D culture systems, including models with and without extracellular matrix (ECM). 3D culture models based on decellularized ECM were discussed in more detail. The role of ECM in pathogeneis of malignant neoplasms, in particular, in the phenomenon of the tumor resistance to chemotherapy, was evaluated. 2D and 3D culture systems were compared, and natural and synthetic ECM were described, as well as the model creation based on 3D bioprinting. Particular attention was paid to in vitro models of various cancers, including those at the metastatic stage, based on 3D cell cultures, which maximally mimic the in vivo tumor behavior. The prospects of the practical application of 3D cell culture models in preclinical drag screening and in personalized therapy were discussed. We also presented our data on in vitro 2D and 3D culturing of human cells on various substrates. 3D cellular models, 3D bioprinting, biotechnology, extracellular matrix, cancer, translational medicine, personalized medicine, drag development, in vitro, ex vivo, oncology The authors are grateful to Dr. E. Shabalina for providing part of the experimental data and to OKA-Biotech Company for the samples of recombinant Funding-The work was supported by a Grant from the Russian Science Foundation (no. 18-15-00391). doi: 10.21519/0234-2758-2020-36-3-3-15
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Betriu, Nausika, Anna Andreeva e Carlos E. Semino. "Erlotinib Promotes Ligand-Induced EGFR Degradation in 3D but Not 2D Cultures of Pancreatic Ductal Adenocarcinoma Cells". Cancers 13, n. 18 (7 settembre 2021): 4504. http://dx.doi.org/10.3390/cancers13184504.
Testo completoAbstract (sommario):
The epithelial growth factor receptor (EGFR) is a tyrosine kinase receptor that participates in many biological processes such as cell proliferation. In addition, EGFR is overexpressed in many epithelial cancers and therefore is a target for cancer therapy. Moreover, EGFR responds to lots of stimuli by internalizing into endosomes from where it can be recycled to the membrane or further sorted into lysosomes where it undergoes degradation. Two-dimensional cell cultures have been classically used to study EGFR trafficking mechanisms in cancer cells. However, it has been widely demonstrated that in 2D cultures cells are exposed to a non-physiological environment as compared to 3D cultures that provide the normal cellular conformation, matrix dimensionality and stiffness, as well as molecular gradients. Therefore, the microenvironment of solid tumors is better recreated in 3D culture models, and this is why they are becoming a more physiological alternative to study cancer physiology. Here, we develop a new model of EGFR internalization and degradation upon erlotinib treatment in pancreatic ductal adenocarcinoma (PDAC) cells cultured in a 3D self-assembling peptide scaffold. In this work, we show that treatment with the tyrosine kinase inhibitor erlotinib promotes EGFR degradation in 3D cultures of PDAC cell lines but not in 2D cultures. We also show that this receptor degradation does not occur in normal fibroblast cells, regardless of culture dimensionality. In conclusion, we demonstrate not only that erlotinib has a distinct effect on tumor and normal cells but also that pancreatic ductal adenocarcinoma cells respond differently to drug treatment when cultured in a 3D microenvironment. This study highlights the importance of culture systems that can more accurately mimic the in vivo tumor physiology.
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Wuchter, Patrick, Rainer Saffrich, Stefan Giselbrecht, Patrick Horn, Anthony D. Ho e Eric Gottwald. "Understanding The Marrow Niche: Advanced 3D Model System Allows Functional Analysis Of The Interaction With Human Hematopoietic Progenitor Cells". Blood 122, n. 21 (15 novembre 2013): 2462. http://dx.doi.org/10.1182/blood.v122.21.2462.2462.
Testo completoAbstract (sommario):
Abstract We previously demonstrated that “stemness” of human hematopoietic progenitor cells (HPC) was maintained in a co-culture setting with a monolayer of human mesenchymal stromal cells (MSC). To simulate and monitor the marrow microenvironment of the HPC niche more precisely we have established a 3D co-culture system based on a proprietary KITChip. The KITChip was developed by the Karlsruhe Institute of Technology (KIT) and represents a unique microchip with defined microwell cavities for 3D cell cultures. Sample acquisition was approved by the local Ethics Committee and informed written consent was obtained from all subjects. MSC were derived from human bone marrow of healthy voluntary donors, and HPC were isolated from umbilical cord blood. Cells were mixed in suspension in a ratio of 3:2 (3x105 MSC and 2x105 HPC) and inoculated into the KITChip, which was subsequently mounted into a microbioreactor. This closed loop setup allowed precise control of medium flow and oxygen saturation. After 1 to 5 days of co-culture, the two cell populations were analyzed by immunostaining, RT2-PCR and colony formation assay. MSC form a complex 3D mesh in the microcavities of the KITChip and were maintained stable for up to 6 weeks. We have demonstrated that HPC were distributed three-dimensionally inside this MSC mesh and could be kept viable in this environment for at least 14 days. A defined proportion of CD34+ HPC adhered to the MSC in the microcavities and built up direct cellular connections to the surrounding MSC. By means of RT2-PCR, we could demonstrate that throughout the whole culture period of 14 days a subpopulation of CD34+/p21+/CXCR4+ cells was maintained in the 3D-environment more efficiently than compared to conventional co-culture with MSC monolayer. This was confirmed by Western blotting after the isolation of both cell populations from the chip. The colony formation assay revealed that the plasticity of the HPC cultivated in the 3D KITChip was nearly the same as that of freshly isolated HPC at day 0, whereas HPC co-cultured on MSC monolayer showed a significant decrease in stem cell plasticity. Further analysis under hypoxic conditions (5% O2) indicated that gene expressions of CD33, CD34, CD38 and CD44 were markedly reduced, while those of CD90, CD105, c-Kit, p21, SDF-1 and Angpt-1 remained stable compared to normoxic culture conditions. This novel model system allows analysis of the major determinants of the niche and the impact of a 3D microenvironment on vital stem cell functions. Early HPC were maintained more efficiently and showed a superior plasticity potential when cultured in the 3D KITChip as compared to conventional 2D co-culture systems. Current studies are in process to define the functional significance of the observed changes in gene expression pattern under hypoxic conditions, which resembles the physiologic milieu of the marrow. Disclosures: Wuchter: ETICHO: Consultancy, Honoraria; Sanofi: Honoraria for lectures Other. Ho:Sanofi-Genzyme: Consultancy, Honoraria, Membership on an entity’s Board of Directors or advisory committees.
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Sanders, Karin, Femke C. A. Ringnalda, Marc L. van de Wetering, Hans S. Kooistra, Björn P. Meij, Hans Clevers e Sara Galac. "Canine Pituitary Organoids as 3D In Vitro Model for Cushing Disease". Journal of the Endocrine Society 5, Supplement_1 (1 maggio 2021): A533. http://dx.doi.org/10.1210/jendso/bvab048.1085.
Testo completoAbstract (sommario):
Abstract Cushing disease (CD) is a serious endocrine disorder that is most often caused by an ACTH-secreting pituitary adenoma. Patients can be treated medically when surgery is not an option or was unsuccessful. However, currently used pituitary-targeting drugs are effective in only 40% of patients. To efficiently identify new pituitary-targeting treatment options, we need an in vitro system that closely mimics in vivo conditions. We therefore aimed to establish organoid cultures of normal anterior pituitary and corticotroph adenomas. Organoids or tumoroids are miniature three-dimensional (3D) structures grown from stem cells, that closely resemble the organ or tumor they originate from. Because CD is a thousand times more prevalent in dogs than in humans, and hypophysectomy is the treatment of choice, we used canine tissues. Normal anterior pituitary glands were collected from three healthy dogs that were euthanized for reasons unrelated to the current study. Corticotroph adenomas were collected from six dogs that underwent transsphenoidal hypophysectomy at our University Clinic. The dogs were diagnosed with CD based on clinical signs, endocrine testing, and CT scan imaging. Normal anterior pituitary and corticotroph adenoma cells were cultured in a 3D matrix (basement membrane extract) with anterior pituitary organoid medium containing specific growth factors and ligands, which was refreshed twice a week. The organoids and tumoroids were characterized with histopathology and RT-qPCR. Structures resembling organoids or tumoroids grew from all nine samples (3 normal, 6 adenoma) that were put in culture. Both cystic and dense structures were observed. The organoids and tumoroids expanded rapidly, and could be passaged once every week. The organoids and tumoroids were successfully cultured up until passage number 10, and were then frozen down. Histopathology showed that the organoid or tumoroid cells morphologically resembled healthy anterior pituitary or corticotroph adenoma cells. All organoids cultures expressed mRNA of pituitary stem cell markers SOX2 and SOX9. This study shows that corticotroph adenomas can be cultured as tumoroids in vitro, something not previously published in any species. Based on the many opportunities in organoid culture (e.g., high-throughput drug screenings, gene editing, studying developmental processes), we expect that this in vitro model will pave the way to efficiently and reliably identify new treatment options for CD. Not only for humans, but also for our best friends: dogs.
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Filipiak-Duliban, Aleksandra, Klaudia Brodaczewska, Arkadiusz Kajdasz e Claudine Kieda. "Spheroid Culture Differentially Affects Cancer Cell Sensitivity to Drugs in Melanoma and RCC Models". International Journal of Molecular Sciences 23, n. 3 (21 gennaio 2022): 1166. http://dx.doi.org/10.3390/ijms23031166.
Testo completoAbstract (sommario):
2D culture as a model for drug testing often turns to be clinically futile. Therefore, 3D cultures (3Ds) show potential to better model responses to drugs observed in vivo. In preliminary studies, using melanoma (B16F10) and renal (RenCa) cancer, we confirmed that 3Ds better mimics the tumor microenvironment. Here, we evaluated how the proposed 3D mode of culture affects tumor cell susceptibility to anti-cancer drugs, which have distinct mechanisms of action (everolimus, doxorubicin, cisplatin). Melanoma spheroids showed higher resistance to all used drugs, as compared to 2D. In an RCC model, such modulation was only observed for doxorubicin treatment. As drug distribution was not affected by the 3D shape, we assessed the expression of MDR1 and mTor. Upregulation of MDR1 in RCC spheroids was observed, in contrast to melanoma. In both models, mTor expression was not affected by the 3D cultures. By NGS, 10 genes related with metabolism of xenobiotics by cytochrome p450 were deregulated in renal cancer spheroids; 9 of them were later confirmed in the melanoma model. The differences between 3D models and classical 2D cultures point to the potential to uncover new non-canonical mechanisms to explain drug resistance set by the tumor in its microenvironment.
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Martin-Iglesias, Sara, Lara Milian, María Sancho-Tello, Rubén Salvador-Clavell, José Javier Martín de Llano, Carmen Carda e Manuel Mata. "BMP-2 Enhances Osteogenic Differentiation of Human Adipose-Derived and Dental Pulp Stem Cells in 2D and 3D In Vitro Models". Stem Cells International 2022 (4 marzo 2022): 1–15. http://dx.doi.org/10.1155/2022/4910399.
Testo completoAbstract (sommario):
Bone tissue provides support and protection to different organs and tissues. Aging and different diseases can cause a decrease in the rate of bone regeneration or incomplete healing; thus, tissue-engineered substitutes can be an acceptable alternative to traditional therapies. In the present work, we have developed an in vitro osteogenic differentiation model based on mesenchymal stem cells (MSCs), to first analyse the influence of the culture media and the origin of the cells on the efficiency of this process and secondly to extrapolate it to a 3D environment to evaluate its possible application in bone regeneration therapies. Two osteogenic culture media were used (one commercial from Stemcell Technologies and a second supplemented with dexamethasone, ascorbic acid, glycerol-2-phosphate, and BMP-2), with human cells of a mesenchymal phenotype from two different origins: adipose tissue (hADSCs) and dental pulp (hDPSCs). The expression of osteogenic markers in 2D cultures was evaluated in several culture periods by means of the immunofluorescence technique and real-time gene expression analysis, taking as reference MG-63 cells of osteogenic origin. The same strategy was extrapolated to a 3D environment of polylactic acid (PLA), with a 3% alginate hydrogel. The expression of osteogenic markers was detected in both hADSCs and hDPSCs, cultured in either 2D or 3D environments. However, the osteogenic differentiation of MSCs was obtained based on the culture medium and the cell origin used, since higher osteogenic marker levels were found when hADSCs were cultured with medium supplemented with BMP-2. Furthermore, the 3D culture used was suitable for cell survival and osteogenic induction.
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Haselager, Marco, Eduard Perelaer, Arnon P. Kater e Eric Eldering. "Development of a Novel Lymph Node-Based 3D Culture System Promoting Chronic Lymphocytic Leukemia Proliferation and Survival". Blood 136, Supplement 1 (5 novembre 2020): 47–48. http://dx.doi.org/10.1182/blood-2020-141962.
Testo completoAbstract (sommario):
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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Kozhina, K. V., E. N. Volkova, I. N. Saburina, Sergey G. Morozov, I. M. Zurina, N. V. Kosheleva, A. A. Gorkun e A. A. Grigorieva. "The influence of peptide bioregulators on skin aging in 3D culture model". Russian Journal of Skin and Venereal Diseases 19, n. 1 (15 febbraio 2016): 58–63. http://dx.doi.org/10.18821/1560-9588-2016-19-1-58-63.
Testo completoAbstract (sommario):
He effect of mesotherapy injection (Meso-Wharton R199TM) on the dermal fibroblasts culture, simulating condition of (mature) aging skin cells are studied. Material and methods. The culture of 4th passage fibroblasts (P4), that corresponds to young skin fibroblasts (control) and the culture of 18th passage fibroblasts (P18), that has all the signs of aging dermal fibroblasts (predominance of large cells, slow cell division) were used. Bioactivity was assessed by cell morphology, epithelium-mesenchyme plasticity and expression of fibroblasts markers: cytokeratin 19, elastin, a-smooth muscle actin (aSMA), PCNA (proliferation marker), collagen types I, III, IV and fibronectin. The formation of spheroids occur when fibroblasts P18 are cultivating with the injection medication, on terms comparable to the formation of spheroids from P4 young fibroblasts. From culture of fibroblasts P18, that was cultured without medication, does not form the full spheroid, but aggregation of cells and their gradual destruction with necrotic masses within the unit are observed. The presence of the medication stimulates the “rejuvenation” of cells and subsequent recovery of the mesenchyme-epithelial plasticity of cultured fibroblasts due to the reduced ability to synthesize sufficient to establish the amount of intercellular contacts the extracellular matrix components (fibronectin and collagen), which affects the ability to form spheroids. Culturing spheroids formed with the medication stimulates expression of elastin, collagen type IV, fibronectin extracellular matrix protein that supports the skin elasticity and superficial cells actively express cytokeratin 19. The study results clearly demonstrate the effectiveness of mesotherapeutic treatment for skin rejuvenation.
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Jähn, K., C. W. Archer, G. Richards e M. J. Stoddart. "A 3D culture model for primary adult human osteoblasts". Bone 44 (maggio 2009): S148—S149. http://dx.doi.org/10.1016/j.bone.2009.01.325.
Testo completo
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Hanasti, Novia, Lia Faridah, Azzania Fibriani, Hesti Lina Wiraswati, Diah Kusumawaty e Savira Ekawardhani. "The Use of Biomaterials in Three-Dimensional Culturing of Cancer Cells". Current Issues in Molecular Biology 45, n. 2 (30 gennaio 2023): 1100–1112. http://dx.doi.org/10.3390/cimb45020073.
Testo completoAbstract (sommario):
Cell culture is an important tool in biological research. Most studies use 2D cell culture, but cells grown in 2D cell culture have drawbacks, including limited cell and cell-extracellular matrix interactions, which make it inaccurate to model conditions in vivo. Anticancer drug screening is an important research and development process for developing new drugs. As an experiment to mimic the cancer environment in vivo, several studies have been carried out on 3-dimensional (3D) cell cultures with added biomaterials. The use of hydrogel in 3D culture cells is currently developing. The type of hydrogel used might influence cell morphology, viability, and drug screening outcome. Therefore, this review discusses 3D cell culture research regarding the addition of biomaterials.
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Petrić, Tina, e Maja Sabol. "Why 3D in vitro cancer models are the future of cancer research?" Periodicum Biologorum 124, n. 3-4 (5 maggio 2023): 69–83. http://dx.doi.org/10.18054/pb.v124i3-4.24697.
Testo completoAbstract (sommario):
Tumors are three-dimensional (3D) entities characterized by complex structural architecture which is necessary for adequate intercellular, intracellular and cell-to-matrix interactions among the aberrant cells in cancer. In the field of cancer research, 2D cell cultures are traditionally used for decades in the majority of experiments. The reasons for this are the vast benefits these models provide, including simplicity and cost effectiveness. However, it is now known that these models are exposed to much higher stiffness, they lose physiological extracellular matrix (ECM) on artificial plastic surfaces as well as differentiation, polarization and cell-cell communication. This leads to the loss of crucial cellular signaling pathways and changes in cell responses to stimuli when compared to in vivo conditions. Moreover, they cannot adequately mimic the complexity and dynamic interactions of the tumor microenvironment (TME) which is of great importance in anticancer drug treatments. 3D models seem more biomimetic compared to 2D cell monolayers because they offer the opportunity to model the cancer mass together with its environment which seems the key factor in promoting and directing cancer invasion. 3D cell culture with its additional dimensionality makes the difference in cellular responses because it influences the spatial and physical aspects of the cells in 3D culture. This affects the signal transduction and makes the behavior of 3D-cultured cells more physiologically relevant and reflective of in vivo cellular responses. This review focuses on major differences between 2D and 3D cell cultures, highlighting the importance of considering bioengineering humanized 3D cancer models as the future in cancer research. Additionally, it presents diverse 3D models currently used in cancer research, outlining their benefits and limitations. Precisely, this review highlights the differences between the 3D models with the focus on tumor stroma interactions, cell population and extracellular matrix composition providing methods and examples for each model from the studies done so far.
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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, n. 13 (23 giugno 2022): 2008. http://dx.doi.org/10.3390/cells11132008.
Testo completoAbstract (sommario):
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.