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BACKGROUND: Cell-scaffold contact measurements are derived from pairs of co-registered volumetric fluorescent confocal laser scanning microscopy (CLSM) images (z-stacks) of stained cells and three types of scaffolds (i.e., spun coat, large microfiber, and medium microfiber). Our analysis of the acquired terabyte-sized collection is motivated by the need to understand the nature of the shape dimensionality (1D vs 2D vs 3D) of cell-scaffold interactions relevant to tissue engineers that grow cells on biomaterial scaffolds. RESULTS: We designed five statistical and three geometrical contact models, and then down-selected them to one from each category using a validation approach based on physically orthogonal measurements to CLSM. The two selected models were applied to 414 z-stacks with three scaffold types and all contact results were visually verified. A planar geometrical model for the spun coat scaffold type was validated from atomic force microscopy images by computing surface roughness of 52.35 nm ±31.76 nm which was 2 to 8 times smaller than the CLSM resolution. A cylindrical model for fiber scaffolds was validated from multi-view 2D scanning electron microscopy (SEM) images. The fiber scaffold segmentation error was assessed by comparing fiber diameters from SEM and CLSM to be between 0.46% to 3.8% of the SEM reference values. For contact verification, we constructed a web-based visual verification system with 414 pairs of images with cells and their segmentation results, and with 4968 movies with animated cell, scaffold, and contact overlays. Based on visual verification by three experts, we report the accuracy of cell segmentation to be 96.4% with 94.3% precision, and the accuracy of cell-scaffold contact for a statistical model to be 62.6% with 76.7% precision and for a geometrical model to be 93.5% with 87.6% precision. CONCLUSIONS: The novelty of our approach lies in (1) representing cell-scaffold contact sites with statistical intensity and geometrical shape models, (2) designing a methodology for validating 3D geometrical contact models and (3) devising a mechanism for visual verification of hundreds of 3D measurements. The raw and processed data are publicly available from https://isg.nist.gov/deepzoomweb/data/ together with the web -based verification system.
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Imageamento Tridimensional/métodos , Modelos Biológicos , Alicerces Teciduais/química , Algoritmos , Materiais Biocompatíveis/química , Células da Medula Óssea/citologia , Humanos , Internet , Masculino , Células-Tronco Mesenquimais/citologia , Microscopia de Força Atômica , Microscopia Confocal , Microscopia Eletrônica de Varredura , Interface Usuário-Computador , Microtomografia por Raio-X , Adulto JovemRESUMO
Cationic nanoparticles (NPs) for targeted gene delivery are conventionally evaluated using 2D in vitro cultures. However, this does not translate well to corresponding in vivo studies because of the marked difference in NP behavior in the presence of the tumor microenvironment. In this study, we investigated whether prostate cancer (PCa) cells cultured in three-dimensional (3D) chitosan-alginate (CA) porous scaffolds could model cationic NP-mediated gene targeted delivery to tumors in vitro. We assessed in vitro tumor cell proliferation, formation of tumor spheroids, and expression of marker genes that promote tumor malignancy in CA scaffolds. The efficacy of NP-targeted gene delivery was evaluated in PCa cells in 2D cultures, PCa tumor spheroids grown in CA scaffolds, and PCa tumors in a mouse TRAMP-C2 flank tumor model. PCa cells cultured in CA scaffolds grew into tumor spheroids and displayed characteristics of higher malignancy as compared to those in 2D cultures. Significantly, targeted gene delivery was only observed in cells cultured in CA scaffolds, whereas cells cultured on 2D plates showed no difference in gene delivery between targeted and nontarget control NPs. In vivo NP evaluation confirmed targeted gene delivery, indicating that only CA scaffolds correctly modeled NP-mediated targeted delivery in vivo. These findings suggest that CA scaffolds serve as a better in vitro platform than 2D cultures for evaluation of NP-mediated targeted gene delivery to PCa.
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Alginatos/química , Quitosana/química , Terapia Genética , Nanopartículas , Neoplasias da Próstata/terapia , Animais , Feminino , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Técnicas In Vitro , Masculino , Camundongos , PorosidadeRESUMO
In the tumor microenvironment, the signals from tumor-associated fibroblasts (TAF) that suppress antitumor immunity remain unclear. Here, we develop and investigate an in vitro three-dimensional (3D) scaffold model for the novel evaluation of TAF interaction with breast tumor cells and breast specific, neu antigen (p98) reactive T cells. Breast cancer cells seeded on 3D chitosan-alginate (CA) scaffolds showed productive growth and formed distinct tumor spheroids. Antigen specific p98 T cells, but not naïve T cells, bound significantly better to tumor cells on scaffolds. The p98 T cells induced potent tumor cell killing but T helper cell cytokine function was impaired in the presence of TAF coseeding on scaffolds. We found that the immunosuppression was mediated, in part, by transforming growth factor beta (TGF-b) and interleukin-10 (IL-10). Therefore, TAF appear capable of inducing potent T cell suppression. CA scaffolds can provide clinically relevant findings prior to preclinical testing of novel immunotherapies.
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Antígenos de Neoplasias/imunologia , Carcinoma/imunologia , Fibroblastos/imunologia , Imunomodulação , Neoplasias Mamárias Animais/imunologia , Alicerces Teciduais , Microambiente Tumoral/imunologia , Alginatos/química , Animais , Antígenos de Neoplasias/genética , Carcinoma/genética , Carcinoma/patologia , Adesão Celular , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Quitosana/química , Técnicas de Cocultura , Feminino , Fibroblastos/patologia , Expressão Gênica , Interleucina-10/imunologia , Interleucina-10/metabolismo , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/patologia , Camundongos , Transdução de Sinais , Linfócitos T/citologia , Linfócitos T/imunologia , Fator de Crescimento Transformador beta/imunologia , Fator de Crescimento Transformador beta/metabolismoRESUMO
PURPOSE: Hepatocellular carcinoma (HCC) is a prevalent solid malignancy. Critically needed discovery of new therapeutics has been hindered by lack of an in vitro cell culture system that can effectively represent the in vivo tumor microenvironment. To address this need, a 3D in vitro HCC model was developed using a biocompatible, chitosan-alginate (CA) scaffold cultured with human HCC cell lines. METHODS: The correlation between the cell function, such as secretion of growth factors and production of ECM in vitro, and the tumor growth and blood vessel recruitment in vivo was investigated. RESULTS: HCC cells grown on 3D CA scaffolds demonstrated morphological characteristics and increased expression of markers of highly malignant cells. Implantation of CA scaffolds cultured with human HCC cells in mice showed accelerated tumor growth. Histology revealed marked differences in morphology and organization of newly formed blood vessels between tumors produced by different pre-cultured conditions. Resistance to doxorubicin was significantly pronounced in CA scaffold-cultured HCC cells compared to 2D or Matrigel cultured HCC cells. CONCLUSIONS: This 3D model of HCC, with its ability to more closely mimic the in vivo tumor behavior, may serve as an invaluable model for study and application of novel anticancer therapeutics against HCC.
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Alginatos/química , Quitosana/química , Resistencia a Medicamentos Antineoplásicos , Neoplasias Hepáticas Experimentais/ultraestrutura , Neovascularização Patológica/patologia , Alicerces Teciduais/química , Ensaios Antitumorais Modelo de Xenoenxerto/métodos , Animais , Antineoplásicos/farmacologia , Materiais Biocompatíveis/química , Técnicas de Cultura de Células/métodos , Linhagem Celular Tumoral , Proliferação de Células/efeitos dos fármacos , Sobrevivência Celular/efeitos dos fármacos , Doxorrubicina/farmacologia , Ácido Glucurônico/química , Ácidos Hexurônicos/química , Humanos , Neoplasias Hepáticas Experimentais/irrigação sanguínea , Neoplasias Hepáticas Experimentais/tratamento farmacológico , Neoplasias Hepáticas Experimentais/metabolismo , Masculino , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Neovascularização Patológica/tratamento farmacológico , Neovascularização Patológica/metabolismoRESUMO
Tissues are organized in hierarchical structures comprised of nanoscale, microscale, and macroscale features. Incorporating hierarchical structures into biomaterial scaffolds may enable better resemblance of native tissue structures and improve cell interaction, but it is challenging to produce such scaffolds using a single conventional scaffold production technique. We developed the Freeze-FRESH (FF) technique that combines FRESH 3D printing (3DP) and freeze-casting to produce 3D printed scaffolds with microscale pores in the struts. FF scaffolds were produced by extrusion 3DP using a support bath at room temperature, followed by freezing and lyophilization, then the FF scaffolds were recovered from the bath and crosslinked. The FF scaffolds had a hierarchical pore structure from the combination of microscale pores throughout the scaffold struts and macroscale pores in the printed design, while control scaffolds had only macroscale pores. FF scaffolds frozen at -20 °C and -80 °C had similar pore sizes, due to freezing in the support bath. The -20 °C and -80 °C FF scaffolds had porous struts with 63.55% ± 2.59% and 56.72% ± 13.17% strut porosity, respectively, while control scaffolds had a strut porosity of 3.15% ± 2.20%. The -20 °C and -80 °C FF scaffolds were softer than control scaffolds: they had pore wall stiffness of 0.17 ± 0.06 MPa and 0.23 ± 0.05 MPa, respectively, compared to 1.31 ± 0.39 MPa for the control. The FF scaffolds had increased resilience in bending compared with control. FF scaffolds supported MDA-MB-231 cell growth and had significantly greater cell numbers than control scaffolds. Cells formed clusters on the porous struts of FF scaffolds and had similar morphologies as the freeze cast scaffolds. The FF technique successfully introduced microscale porosity into the 3DP scaffold struts to produce hierarchical pore structures that enhanced MDA-MB-231 growth.
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Prostate cancer (PCa) is a common cancer in men that is curable prior to metastasis, when its prognosis worsens. Chondroitin sulfate (CS) is found in the extracellular matrix of normal prostate tissue and PCa, with greater content in metastatic PCa. Biomaterial scaffolds containing CS have yet to be evaluated for tumor microenvironment applications. Three-dimensional porous chitosan-CS (C-CS) scaffolds were developed and evaluated for PCa culture. Three C-CS scaffold compositions were prepared with 4 w/v% chitosan and 0.1, 0.5, and 1.0 w/v% CS and named 4-0.1, 4-0.5, and 4-1, respectively. The C-CS scaffolds had 90-95% porosity, average pore sizes between 143 and 166 µm, and no significant difference in scaffold stiffness. PC-3 and 22Rv1 PCa cells were cultured on the C-CS scaffolds to study the effect of CS on PCa growth and epithelial to mesenchymal transition (EMT). All C-CS scaffold compositions supported PCa growth and the 4-1 scaffolds had the greatest cell numbers for both PC-3 and 22Rv1. The C-CS scaffolds promoted upregulated EMT marker expression compared to 2D cultures with the greatest EMT marker expression in 4-1 scaffolds. Increasing CS concentration promoted upregulated vimentin expression in PC-3 cultures and N-cadherin and MMP-2 expression in 22Rv1 cultures. C-CS scaffolds promoted docetaxel drug resistance in PC-3 and 22Rv1 cultures and the 4-1 scaffold cultures had the greatest drug resistance. These results indicate that C-CS scaffolds are a promising in vitro platform for PCa.
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Quitosana , Neoplasias da Próstata , Proliferação de Células , Sulfatos de Condroitina , Transição Epitelial-Mesenquimal , Humanos , Masculino , Porosidade , Alicerces Teciduais , Microambiente TumoralRESUMO
This study investigated a mechanical processing method using vibratory milling to reduce the particle size of bone ash to produce hydroxyapatite (HA) nanoparticles from bovine bone. Bovine femurs were cleaned of soft tissue, cut into small pieces, heated to 600 deg for 24 h, and ground into a coarse powder. A 50 wt% suspension was prepared, vibratory milled for 18 h, and then the milled suspension was filter pressed, dried, and ground into powder. The powder was analyzed with scanning electron microscopy and X-ray diffraction, which confirmed the particle size and the chemical composition of the powder-matched HA. The results of this study qualitatively showed that it is possible to produce HA nanoparticles from bone. This research explores a "green" manufacturing process that reuses a scrap material from the food industry and reduces the use of chemical precursors for synthetic nanoparticle synthesis. Such HA powder can potentially be used as a bone substitute and for coating orthopedic and dental implants.
Assuntos
Durapatita/análise , Fêmur/química , Nanopartículas , Animais , Bovinos , Microscopia Eletrônica de Varredura , Vibração , Difração de Raios XRESUMO
Prostate cancer (PCa) is a leading cause of death for men worldwide. Most PCa patients die from metastasis and bone is the most common metastatic site. Three dimensional (3D) porous chitosan-alginate (CA) scaffolds were developed for bone tissue engineering and demonstrated for culture of cancer cells and enrichment of cancer stem cells. However, only a single scaffold composition was studied. Three compositions of 3D porous CA scaffolds (2, 4, and 6â¯wt%) were used to investigate the effect of scaffold stiffness on PCa cell response with PC-3, C4-2B, and 22Rv1 cell lines. The PC-3â¯cells formed cell clusters while the C4-2B and 22Rv1 cells formed multicellular spheroids. The three cell lines demonstrated stiffness independent cell growth and expressed phenotypic PCa biomarkers. The osteoblastic PCa lines C4-2B and 22Rv1 mineralized in basal media, while the osteolytic PC-3 line did not, demonstrating that CA scaffold cultures revealed differences in PCa phenotypes. The CA scaffolds are a 3D culture platform that supports PCa growth and phenotypic expression with adjustable scaffold stiffness to mimic stages of metastatic progression. Further investigation of the scaffolds for co-culture of PCa cells with fibroblasts and primary PCa cell culture should be conducted to develop a platform for screening chemotherapies.
Assuntos
Alginatos/química , Quitosana/química , Neoplasias da Próstata/patologia , Alicerces Teciduais/química , Actinas/metabolismo , Caderinas/metabolismo , Calcificação Fisiológica , Comunicação Celular , Linhagem Celular Tumoral , Proliferação de Células , Forma Celular , Regulação Neoplásica da Expressão Gênica , Humanos , Masculino , Proteínas de Neoplasias/genética , Proteínas de Neoplasias/metabolismo , Osteoblastos/metabolismo , Osteocalcina/metabolismo , Fenótipo , Porosidade , Neoplasias da Próstata/genética , Neoplasias da Próstata/ultraestruturaRESUMO
In living systems, it is frequently stated that form follows function by virtue of evolutionary pressures on organism development, but in the study of how functions emerge at the cellular level, function often follows form. We study this chicken versus egg problem of emergent structure-property relationships in living systems in the context of primary human bone marrow stromal cells cultured in a variety of microenvironments that have been shown to cause distinct patterns of cell function and differentiation. Through analysis of a publicly available catalog of three-dimensional (3D) cell shape data, we introduce a family of metrics to characterize the 'form' of the cell populations that emerge from a variety of diverse microenvironments. In particular, measures of form are considered that are expected to have direct significance for cell function, signaling and metabolic activity: dimensionality, polarizability and capacitance. Dimensionality was assessed by an intrinsic measure of cell shape obtained from the polarizability tensor. This tensor defines ellipsoids for arbitrary cell shapes and the thinnest dimension of these ellipsoids, P 1, defines a reference minimal scale for cells cultured in a 3D microenvironment. Polarizability governs the electric field generated by a cell, and determines the cell's ability to detect electric fields. Capacitance controls the shape dependence of the rate at which diffusing molecules contact the surface of the cell, and this has great significance for inter-cellular signaling. These results invite new approaches for designing scaffolds which explicitly direct cell dimensionality, polarizability and capacitance to guide the emergence of new cell functions derived from the acquired form.
Assuntos
Técnicas de Cultura de Células/métodos , Diferenciação Celular/efeitos dos fármacos , Microambiente Celular , Células-Tronco Mesenquimais/citologia , Alicerces Teciduais/química , Algoritmos , Animais , Núcleo Celular/metabolismo , Forma Celular , Eletricidade , Fibrinogênio/química , Humanos , Camundongos , Microscopia Confocal , Nanofibras/química , Poliestirenos/química , Probabilidade , Transdução de Sinais , Trombina/químicaRESUMO
Nanotechnology is a rapidly developing area in science involved with manipulating matter at the atomic or molecular level. Nanotechnology is typically defined at a scale on the order of less than approximately 100 nm. Matter possesses unique properties at these size levels that are neither Newtonian nor quantum, but between the two regimes.These unique properties have created significant interest and excitement, sparking numerous research investigations. Nanotechnology is a very broad field with many current and potential applications. Some important examples of applications include battlefield activated dynamic armor clothing for soldiers, additives to sunscreens, and diagnostic laboratories on a chip to monitor general personal health. Groundbreaking capabilities often raise new questions. Any new scientific or technological development has the usual concomitant associated ethical issues, specifically regarding containment and regulation. These ethical issues are more pronounced with nanotechnology due to the sharp divide between those who see its great potential and opponents who express fears. Nanotechnology supporters believe that it has the potential to transform our lives dramatically, while opponents of nanotechnology fear that self-replicating "nanobots" could escape from laboratories and reduce all life on earth to "gray goo. "These fears have swayed generally uninformed public opinions via the media and sensational entertainment. A critical discussion of ethical issues surrounding nanotechnology, including the interaction of nanotechnology with the body and the environment--nanobiotechnology--and regulation of nanotechnology, is presented. We advocate strong, uniform regulations for nanotechnology, but only the use of regulations as needed. The limited use of regulations prevents the regulations from becoming burdensome and inhibiting research in the field.
Assuntos
Biotecnologia/ética , Nanotecnologia/ética , Humanos , Nanotecnologia/tendênciasRESUMO
Periodically patterned Au nanorods in TiO2 nanocavities (Au NRs@TiO2) were fabricated via magnetron sputtering followed by a thermal dewetting process. This innovative Au NRs@TiO2 heterostructure was used as a plasmonic sensing platform for photoelectrochemical detection of glucose and lactose. This Au NRs@TiO2 patterned heterostructure possesses superior sensing properties to other Au nanoparticle-based sensors because (i) localized surface plasmon resonance (LSPR) generated at Au/TiO2 interfaces enhanced sensitivity of glucose (lactose) amperometric detection; (ii) periodic Au nanocrystals in TiO2 nanocavities accelerated charge separation and transfer rate, especially under monochromatic blue light irradiation; (iii) discrete planar architectures comprising Au NRs immobilized on TiO2 substrates significantly improved stability and reusability of the sensors. A low detection limit of 1 µM (10 µM) and a high sensitivity of 812 µA mM-1 cm-2 (270 µA mM-1 cm-2) were achieved on the Au NRs@TiO2 heterostructures for glucose (lactose) detection without the addition of enzymes. Good selectivity and superb stability over more than 8 weeks was also demonstrated using these Au NRs@TiO2 heterostructures for glucose (lactose) detection. Additionally, this cost-efficient technique can be easily extended to other photoelectrochemical sensing systems when considering the combination of sensing and visible or infrared light source enhancement.
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Many biomaterial scaffolds have been advanced to provide synthetic cell niches for tissue engineering and drug screening applications; however, current methods for comparing scaffold niches focus on cell functional outcomes or attempt to normalize materials properties between different scaffold formats. We demonstrate a three-dimensional (3D) cellular morphotyping strategy for comparing biomaterial scaffold cell niches between different biomaterial scaffold formats. Primary human bone marrow stromal cells (hBMSCs) were cultured on 8 different biomaterial scaffolds, including fibrous scaffolds, hydrogels, and porous sponges, in 10 treatment groups to compare a variety of biomaterial scaffolds and cell morphologies. A bioinformatics approach was used to determine the 3D cellular morphotype for each treatment group by using 82 shape metrics to analyze approximately 1000 cells. We found that hBMSCs cultured on planar substrates yielded planar cell morphotypes, while those cultured in 3D scaffolds had elongated or equiaxial cellular morphotypes with greater height. Multivariate analysis was effective at distinguishing mean shapes of cells in flat substrates from cells in scaffolds, as was the metric L1-depth (the cell height along its shortest axis after aligning cells with a characteristic ellipsoid). The 3D cellular morphotyping technique enables direct comparison of cellular microenvironments between widely different types of scaffolds and design of scaffolds based on cell structure-function relationships.
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Cancer stem cells are increasingly becoming a primary target for new cancer treatment development. The ability to study their transient behavior in vitro will provide the opportunity for high-throughput testing of more effective therapies. We have previously demonstrated the use of 3D porous chitosan-alginate (CA) scaffolds to promote cancer stem-like cell (CSC) proliferation and enrichment in glioblastoma. Here we use 3D porous CA scaffolds to promote cancer stem-like cell enrichment in cell lines from prostate, liver, and breast cancers, and investigate the proliferation, morphology, and gene expressions of cells cultured in CA scaffolds as compared to 2D controls. The 3D CA scaffold cultures for all three cancer types showed reduced proliferation, formation of tumor spheroids, and increased expression of CSC associated mark genes (CD133 and NANOG), as opposed to monolayers. Additionally, we present a putative mechanism for the cancer stem-like cell enrichment on CA scaffolds. This study demonstrates that the cancer stem-like cell enrichment in CA scaffolds is a robust process that is not restricted to particular cancer types.
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Cell morphology has been identified as a potential indicator of stem cell response to biomaterials. However, determination of cell shape phenotype in biomaterials is complicated by heterogeneous cell populations, microenvironment heterogeneity, and multi-parametric definitions of cell morphology. To associate cell morphology with cell-material interactions, we developed a shape phenotyping framework based on support vector machines. A feature selection procedure was implemented to select the most significant combination of cell shape metrics to build classifiers with both accuracy and stability to identify and predict microenvironment-driven morphological differences in heterogeneous cell populations. The analysis was conducted at a multi-cell level, where a "supercell" method used average shape measurements of small groups of single cells to account for heterogeneous populations and microenvironment. A subsampling validation algorithm revealed the range of supercell sizes and sample sizes needed for classifier stability and generalization capability. As an example, the responses of human bone marrow stromal cells (hBMSCs) to fibrous vs flat microenvironments were compared on day 1. Our analysis showed that 57 cells (grouped into supercells of size 4) are the minimum needed for phenotyping. The analysis identified that a combination of minor axis length, solidity, and mean negative curvature were the strongest early shape-based indicator of hBMSCs response to fibrous microenvironment.
Assuntos
Tamanho Celular , Microambiente Celular/fisiologia , Aprendizado de Máquina , Mecanotransdução Celular/fisiologia , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/fisiologia , Microscopia/métodos , Células Cultivadas , Humanos , Interpretação de Imagem Assistida por Computador/métodos , Reconhecimento Automatizado de Padrão/métodos , FenótipoRESUMO
Effective elicitation of endogenous immunity is associated with improved prognosis for cancer patients. Clinical evidence in hematological and solid cancers shows that intratumoral injection of immunostimulatory genes primes and augments endogenous T cell responses. The ability of pro-inflammatory chemokines/cytokines to facilitate migration/activation of antigen-presenting cells (APC) and lymphocytes prompted our modeling of intratumoral delivery of a chemokine/cytokine combination for breast cancer treatment. Here, we demonstrate that expression of chemokine ligand 21 (CCL21) and interferon gamma (IFNγ) in tumors improves tumor specific T cell recruitment to tumor and activation in the tumor milieu. IFNγ and CCL21 were delivered into tumor cells via plasmids, and transfected cells were seeded to form spheroids on three-dimensional (3D) chitosan-alginate (CA) scaffolds. Co-expression of CCL21 and IFNγ, as evidenced by qRT-PCR and ELISA, induced increased recruitment, binding, and infiltration of anti-neu (p98) peptide specific T cells into the breast tumors as determined by SEM and immunofluorescence assays. The co-expression promoted recruitment of only p98 T cells, but not naïve T cells, demonstrating an antigen-restricted activation. Furthermore, the co-expression impacted T helper (Th) cell immunity, promoting an increase in secretion of pro-inflammatory Th-associated cytokine, tumor necrosis factor alpha (TNFα), and cytotoxic T lymphocyte (CTL)-associated protease, Granzyme B (GzB). Therefore, 3D CA scaffolds may be a useful breast cancer tumor microenvironment model to evaluate T cell function. Further characterization of CCL21-IFNγ mediated anti-tumor immunity will potentially benefit the development of chemokine/cytokine combination platforms as anti-cancer agents.
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Quimiocina CCL21/metabolismo , Interferon gama/metabolismo , Neoplasias Mamárias Animais/imunologia , Esferoides Celulares/imunologia , Linfócitos T Citotóxicos/metabolismo , Linfócitos T Auxiliares-Indutores/metabolismo , Alginatos , Animais , Antígenos de Neoplasias/imunologia , Antígenos de Neoplasias/metabolismo , Linhagem Celular Tumoral , Movimento Celular , Quimiocina CCL21/genética , Quimiocina CCL21/imunologia , Quitosana , Feminino , Ácido Glucurônico , Granzimas/metabolismo , Ácidos Hexurônicos , Imunidade Celular , Interferon gama/genética , Interferon gama/imunologia , Neoplasias Mamárias Animais/genética , Neoplasias Mamárias Animais/metabolismo , Neoplasias Mamárias Animais/patologia , Camundongos , Esferoides Celulares/metabolismo , Esferoides Celulares/patologia , Linfócitos T Citotóxicos/imunologia , Linfócitos T Auxiliares-Indutores/imunologia , Microambiente TumoralRESUMO
Emerging evidence implicates cancer stem cells (CSCs) as primary determinants of the clinical behavior of human cancers, representing an ideal target for next-generation anti-cancer therapies. However CSCs are difficult to propagate in vitro, severely limiting the study of CSC biology and drug development. Here we report that growing cells from glioblastoma (GBM) cell lines on three dimensional (3D) porous chitosan-alginate (CA) scaffolds dramatically promotes the proliferation and enrichment of cells possessing the hallmarks of CSCs. CA scaffold-grown cells were found more tumorigenic in nude mouse xenografts than cells grown from monolayers. Growing in CA scaffolds rapidly promoted expression of genes involved in the epithelial-to-mesenchymal transition that has been implicated in the genesis of CSCs. Our results indicate that CA scaffolds have utility as a simple and inexpensive means to cultivate CSCs in vitro in support of studies to understand CSC biology and develop more effective anti-cancer therapies.
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Alginatos/farmacologia , Proliferação de Células/efeitos dos fármacos , Quitosana/farmacologia , Células-Tronco Neoplásicas/efeitos dos fármacos , Alicerces Teciduais/química , Alginatos/química , Animais , Antineoplásicos/química , Antineoplásicos/farmacologia , Linhagem Celular Tumoral , Quitosana/química , Transição Epitelial-Mesenquimal/efeitos dos fármacos , Glioblastoma , Ácido Glucurônico/química , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/química , Ácidos Hexurônicos/farmacologia , Humanos , Camundongos , Camundongos Nus , Microscopia Eletrônica de Varredura , Células-Tronco Neoplásicas/citologia , Ensaios Antitumorais Modelo de XenoenxertoRESUMO
This study investigated the use of three-dimensional porous chitosan-alginate (CA) scaffolds for critical size calvarial defect (diameter, 5.0 mm) repair in Sprague-Dawley rats. CA scaffolds have been used for in vitro culture of many cell types and demonstrated osteogenesis in ectopic locations in vivo, but have yet to be evaluated for functional bone tissue engineering applications. CA scaffolds demonstrated the ability to support undifferentiated mesenchymal stem cells (MSCs) in culture for 14 days in vitro and promoted spherical morphology. In vivo tests were performed using CA scaffolds and CA scaffolds with treatments including undifferentiated MSCs, bone marrow aspirate, and bone morphogenetic protein-2 (BMP-2) growth factor in comparison to unfilled bone defect used as a control. The samples were analyzed with MicroCT, histology, and immunohistochemical staining at 4 and 16 weeks. Partial defect closure was observed in all experimental groups at 16 weeks, with the greatest defect closure (71.56 ± 19.74%) in the animal group treated with CA scaffolds with BMP-2 (CA + BMP-2). The experimental samples demonstrated osteogenesis in histology and immunohistochemical staining, with the CA + BMP-2 group, showing the greatest level of osteogenesis. Tissue engineered CA scaffolds show promise in reconstruction of critical size bone defects.
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Alginatos/farmacologia , Regeneração Óssea/efeitos dos fármacos , Quitosana/farmacologia , Crânio/efeitos dos fármacos , Crânio/patologia , Alicerces Teciduais/química , Animais , Células Cultivadas , Ácido Glucurônico/farmacologia , Ácidos Hexurônicos/farmacologia , Processamento de Imagem Assistida por Computador , Células-Tronco Mesenquimais/citologia , Células-Tronco Mesenquimais/efeitos dos fármacos , Células-Tronco Mesenquimais/ultraestrutura , Osteocalcina/metabolismo , Osteopontina/metabolismo , Implantação de Prótese , Ratos , Ratos Sprague-Dawley , Crânio/diagnóstico por imagem , Cicatrização/efeitos dos fármacos , Microtomografia por Raio-XRESUMO
Cancer therapeutics are developed through extensive screening; however, many therapeutics evaluated with 2D in vitro cultures during pre-clinical trials suffer from lower efficacy in patients. Replicating the in vivo tumor microenvironment in vitro with three-dimensional (3D) porous scaffolds offers the possibility of generating more predictive pre-clinical models to enhance cancer treatment efficacy. We developed a chitosan and hyaluronic acid (HA) polyelectrolyte complex 3D porous scaffold and evaluated its physical properties. Chitosan-HA (C-HA) scaffolds had a highly porous network. C-HA scaffolds were compared to 2D surfaces for in vitro culture of U-118 MG human glioblastoma (GBM) cells. C-HA scaffold cultures promoted tumor spheroid formation and increased stem-like properties of GBM cells as evidenced by the upregulation of CD44, Nestin, Musashi-1, GFAP, and HIF-1α as compared with 2D cultures. Additionally, the invasiveness of GBM cells cultured in C-HA scaffolds was significantly enhanced compared to those grown in 2D cultures. C-HA scaffold cultures were also more resistant to chemotherapy drugs, which corresponded to the increased expression of ABCG2 drug efflux transporter. These findings suggest that C-HA scaffolds offer promise as an in vitro GBM platform for study and screening of novel cancer therapeutics.
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Quitosana/química , Matriz Extracelular/metabolismo , Glioblastoma/metabolismo , Ácido Hialurônico/química , Alicerces Teciduais/química , Linhagem Celular Tumoral , Proliferação de Células , Humanos , Microscopia Eletrônica de Varredura , Porosidade , Espectroscopia de Infravermelho com Transformada de FourierRESUMO
Osteochondral tissue engineering poses the challenge of combining both cartilage and bone tissue engineering fundamentals. In this study, a sphere-templating technique was applied to fabricate an integrated bi-layered scaffold based on degradable poly(hydroxyethyl methacrylate) hydrogel. One layer of the integrated scaffold was designed with a single defined, monodispersed pore size of 38 µm and pore surfaces coated with hydroxyapatite particles to promote regrowth of subchondral bone while the second layer had 200 µm pores with surfaces decorated with hyaluronan for articular cartilage regeneration. Mechanical properties of the construct as well as cyto-compatibility of the scaffold and its degradation products were elucidated. To examine the potential of the biphasic scaffold for regeneration of osteochondral tissue the designated cartilage and bone layers of the integrated bi-layered scaffold were seeded with chondrocytes differentiated from human mesenchymal stem cells and primary human mesenchymal stem cells, respectively. Both types of cells were co-cultured within the scaffold in standard medium without soluble growth/differentiation factors over four weeks. The ability of the integrated bi-layered scaffold to support simultaneous matrix deposition and adequate cell growth of two distinct cell lineages in each layer during four weeks of co-culture in vitro in the absence of soluble growth factors was demonstrated.
Assuntos
Condrócitos/citologia , Condrogênese/fisiologia , Células-Tronco Mesenquimais/citologia , Osteoblastos/citologia , Osteogênese/fisiologia , Engenharia Tecidual/instrumentação , Alicerces Teciduais , Células Cultivadas , Condrócitos/fisiologia , Desenho de Equipamento , Análise de Falha de Equipamento , Humanos , Células-Tronco Mesenquimais/fisiologia , Osteoblastos/fisiologia , Integração de SistemasRESUMO
The treatment of castration-resistant prostate cancer (CRPC) remains palliative. Immunotherapy offers a potentially effective therapy for CRPC; however, its advancement into the clinic has been slow, in part because of the lack of representative in vitro tumor models that resemble the in vivo tumor microenvironment for studying interactions of CRPC cells with immune cells and other potential therapeutics. This study evaluates the use of 3D porous chitosan-alginate (CA) scaffolds for culturing human prostate cancer (PCa) cells and studying tumor cell interaction with human peripheral blood lymphocytes (PBLs) ex vivo. CA scaffolds and Matrigel matrix samples support in vitro tumor spheroid formation over 15 d of culture, and CA scaffolds support live-cell fluorescence imaging with confocal microscopy using stably transfected PCa cells for 55 d. PCa cells grown in Matrigel matrix and CA scaffolds for 15 d are co-cultured with PBLs for 2 and 6 d in vitro and evaluated with scanning electron microscopy (SEM), immunohistochemistry (IHC), and flow cytometry. Both the Matrigel matrix and CA scaffolds support interaction of PBLs with PCa tumors, with CA scaffolds providing a more robust platform for subsequent analyses. This study demonstrates the use of 3D natural polymer scaffolds as a tissue culture model for supporting long-term analysis of interaction of prostate cancer tumor cells with immune cells, providing an in vitro platform for rapid immunotherapy development.