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      Development of a Dynamic Stem Cell Culture Platform for Mesenchymal Stem Cell Adhesion and Evaluation

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          Abstract

          The importance of providing a physiologically relevant environment for cell culture is well recognized. The combination of proper environmental cues which are provided in vivo by the bloodstream and extracellular matrix must be reproduced to properly examine cell response in vitro, and cannot be recapitulated using traditional culture on polystyrene. Here, we have developed a device, the dynamic stem cell culture platform (DSCCP), consisting of a biomimetic scaffold cultured within the dynamic environment of a perfusion bioreactor. By varying scaffold parameters including stiffness and protein inclusion at the material surface, we found that human mesenchymal stem cells (hMSCs) were able to adhere to modified substrates, while still maintaining multipotency. Culture in a perfusion bioreactor showed cell survival and proliferation, particularly on modified substrates. The DSCCP represents a complete platform for cell adhesion and subsequent evaluation, including the response of a cell population to drug treatment.

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          Most cited references25

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          Tensional homeostasis and the malignant phenotype.

          Tumors are stiffer than normal tissue, and tumors have altered integrins. Because integrins are mechanotransducers that regulate cell fate, we asked whether tissue stiffness could promote malignant behavior by modulating integrins. We found that tumors are rigid because they have a stiff stroma and elevated Rho-dependent cytoskeletal tension that drives focal adhesions, disrupts adherens junctions, perturbs tissue polarity, enhances growth, and hinders lumen formation. Matrix stiffness perturbs epithelial morphogenesis by clustering integrins to enhance ERK activation and increase ROCK-generated contractility and focal adhesions. Contractile, EGF-transformed epithelia with elevated ERK and Rho activity could be phenotypically reverted to tissues lacking focal adhesions if Rho-generated contractility or ERK activity was decreased. Thus, ERK and Rho constitute part of an integrated mechanoregulatory circuit linking matrix stiffness to cytoskeletal tension through integrins to regulate tissue phenotype.
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            Capturing complex 3D tissue physiology in vitro.

            The emergence of tissue engineering raises new possibilities for the study of complex physiological and pathophysiological processes in vitro. Many tools are now available to create 3D tissue models in vitro, but the blueprints for what to make have been slower to arrive. We discuss here some of the 'design principles' for recreating the interwoven set of biochemical and mechanical cues in the cellular microenvironment, and the methods for implementing them. We emphasize applications that involve epithelial tissues for which 3D models could explain mechanisms of disease or aid in drug development.
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              Migration of tumor cells in 3D matrices is governed by matrix stiffness along with cell-matrix adhesion and proteolysis.

              Cell migration on 2D surfaces is governed by a balance between counteracting tractile and adhesion forces. Although biochemical factors such as adhesion receptor and ligand concentration and binding, signaling through cell adhesion complexes, and cytoskeletal structure assembly/disassembly have been studied in detail in a 2D context, the critical biochemical and biophysical parameters that affect cell migration in 3D matrices have not been quantitatively investigated. We demonstrate that, in addition to adhesion and tractile forces, matrix stiffness is a key factor that influences cell movement in 3D. Cell migration assays in which Matrigel density, fibronectin concentration, and beta1 integrin binding are systematically varied show that at a specific Matrigel density the migration speed of DU-145 human prostate carcinoma cells is a balance between tractile and adhesion forces. However, when biochemical parameters such as matrix ligand and cell integrin receptor levels are held constant, maximal cell movement shifts to matrices exhibiting lesser stiffness. This behavior contradicts current 2D models but is predicted by a recent force-based computational model of cell movement in a 3D matrix. As expected, this 3D motility through an extracellular environment of pore size much smaller than cellular dimensions does depend on proteolytic activity as broad-spectrum matrix metalloproteinase (MMP) inhibitors limit the migration of DU-145 cells and also HT-1080 fibrosarcoma cells. Our experimental findings here represent, to our knowledge, discovery of a previously undescribed set of balances of cell and matrix properties that govern the ability of tumor cells to migration in 3D environments.
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                Author and article information

                Journal
                Mol Pharm
                Mol. Pharm
                mp
                mpohbp
                Molecular Pharmaceutics
                American Chemical Society
                1543-8384
                1543-8392
                12 March 2015
                12 March 2014
                07 July 2014
                : 11
                : 7 , Engineered Biomimetic Tissue Platforms for in Vitro Drug Evaluation
                : 2172-2181
                Affiliations
                []Fischell Department of Bioengineering, University of Maryland , College Park, Maryland 20742, United States
                []FDA, CDRH , Silver Spring, Maryland 20993, United States
                Author notes
                [* ]Fischell Department of Bioengineering, University of Maryland, 3238 Jeong H. Kim Engineering Building (#225), College Park, Maryland 20742. Tel: 301 405 7475. Fax: 301 405 0523. E-mail: jpfisher@ 123456umd.edu . Web: http://www.glue.umd.edu/∼jpfisher.
                Article
                10.1021/mp500062n
                4086736
                24620713
                6ba78c48-ba4c-4a5b-8fc0-c791fd941aa4
                Copyright © 2014 American Chemical Society

                Terms of Use

                History
                : 20 January 2014
                : 12 March 2014
                : 06 March 2014
                Funding
                National Institutes of Health, United States
                Categories
                Article
                Custom metadata
                mp500062n
                mp-2014-00062n

                Pharmacology & Pharmaceutical medicine
                stem cells,cell adhesion,hydrogel,bioreactor
                Pharmacology & Pharmaceutical medicine
                stem cells, cell adhesion, hydrogel, bioreactor

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