Browsing by Author "Tseng, Hubert"

Now showing 1 - 7 of 7
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    A high-throughput three-dimensional cell migration assay for toxicity screening with mobile device-based macroscopic image analysis
    (Nature Publishing Group, 2013) Timm, David M.; Chen, Jianbo; Sing, David; Gage, Jacob A.; Haisler, William L.; Neeley, Shane K.; Raphael, Robert M.; Dehghani, Mehdi; Rosenblatt, Kevin P.; Killian, T.C.; Tseng, Hubert; Souza, Glauco R.
    There is a growing demand for in vitro assays for toxicity screening in three-dimensional (3D) environments. In this study, 3D cell culture using magnetic levitation was used to create an assay in which cells were patterned into 3D rings that close over time. The rate of closure was determined from time-lapse images taken with a mobile device and related to drug concentration. Rings of human embryonic kidney cells (HEK293) and tracheal smooth muscle cells (SMCs) were tested with ibuprofen and sodium dodecyl sulfate (SDS). Ring closure correlated with the viability and migration of cells in two dimensions (2D). Images taken using a mobile device were similar in analysis to images taken with a microscope. Ring closure may serve as a promising label-free and quantitative assay for high-throughput in vivo toxicity in 3D cultures.
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    A spheroid toxicity assay using magnetic 3D bioprinting and real-time mobile device-based imaging
    (Springer Nature, 2015) Tseng, Hubert; Gage, Jacob A.; Shen, Tsaiwei; Haisler, William L.; Neeley, Shane K.; Shiao, Sue; Chen, Jianbo; Desai, Pujan K.; Liao, Angela; Hebel, Chris; Raphael, Robert M.; Becker, Jeanne L.; Souza, Glauco R.
    An ongoing challenge in biomedical research is the search for simple, yet robust assays using 3D cell cultures for toxicity screening. This study addresses that challenge with a novel spheroid assay, wherein spheroids, formed by magnetic 3D bioprinting, contract immediately as cells rearrange and compact the spheroid in relation to viability and cytoskeletal organization. Thus, spheroid size can be used as a simple metric for toxicity. The goal of this study was to validate spheroid contraction as a cytotoxic endpoint using 3T3 fibroblasts in response to 5 toxic compounds (all-trans retinoic acid, dexamethasone, doxorubicin, 5′-fluorouracil, forskolin), sodium dodecyl sulfate (+control), and penicillin-G (−control). Real-time imaging was performed with a mobile device to increase throughput and efficiency. All compounds but penicillin-G significantly slowed contraction in a dose-dependent manner (Z’ = 0.88). Cells in 3D were more resistant to toxicity than cells in 2D, whose toxicity was measured by the MTT assay. Fluorescent staining and gene expression profiling of spheroids confirmed these findings. The results of this study validate spheroid contraction within this assay as an easy, biologically relevant endpoint for high-throughput compound screening in representative 3D environments.
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    Assembly of a Three-Dimensional Multitype Bronchiole Coculture Model Using Magnetic Levitation
    (Liebert, 2013) Tseng, Hubert; Gage, Jacob A.; Raphael, Robert M.; Moore, Robert H.; Killian, Thomas C.; Grande-Allen, K. Jane; Souza, Glauco R.
    A longstanding goal in biomedical research has been to create organotypic cocultures that faithfully represent native tissue environments. There is presently great interest in representative culture models of the lung, which is a particularly challenging tissue to recreate in vitro. This study used magnetic levitation in conjunction with magnetic nanoparticles as a means of creating an organized three-dimensional (3D) coculture of the bronchiole that sequentially layers cells in a manner similar to native tissue architecture. The 3D coculture model was assembled from four human cell types in the bronchiole: endothelial cells, smooth muscle cells (SMCs), fibroblasts, and epithelial cells (EpiCs). This study represents the first effort to combine these particular cell types into an organized bronchiole coculture. These cell layers were first cultured in 3D by magnetic levitation, and then manipulated into contact with a custom-made magnetic pen, and again cultured for 48 h. Hematoxylin and eosin staining of the resulting coculture showed four distinct layers within the 3D coculture. Immunohistochemistry confirmed the phenotype of each of the four cell types and showed organized extracellular matrix formation, particularly, with collagen type I. Positive stains for CD31, von Willebrand factor, smooth muscle a-actin, vimentin, and fibronectin demonstrate the maintenance of the phenotype for endothelial cells, SMCs, and fibroblasts. Positive stains for mucin-5AC, cytokeratin, and E-cadherin after 7 days with and without 1% fetal bovine serum showed that EpiCs maintained the phenotype and function. This study validates magnetic levitation as a method for the rapid creation of organized 3D cocultures that maintain the phenotype and induce extracellular matrix formation.
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    Fabrication and Mechanical Evaluation of Anatomically-Inspired Quasilaminate Hydrogel Structures with Layer-Specific Formulations
    (Springer, 2012) Tseng, Hubert; Cuchiara, Maude L.; Durst, Christopher A.; Cuchiara, Michael P.; Lin, Chris J.; West, Jennifer L.; Grande-Allen, K. Jane
    A major tissue engineering challenge is the creation of multilaminate scaffolds with layer-specific mechanical properties representative of native tissues, such as heart valve leaflets, blood vessels, and cartilage. For this purpose, poly(ethylene glycol) diacrylate (PEGDA) hydrogels are attractive materials due to their tunable mechanical and biological properties. This study explored the fabrication of trilayer hydrogel quasilaminates. A novel sandwich method was devised to create quasilaminates with layers of varying stiffnesses. The trilayer structure was comprised of two �stiff� outer layers and one �soft� inner layer. Tensile testing of bilayer quasilaminates demonstrated that these scaffolds do not fail at the interface. Flexural testing showed that the bending modulus of acellular quasilaminates fell between the bending moduli of the �stiff� and �soft� hydrogel layers. The bending modulus and swelling of trilayer scaffolds with the same formulations were not significantly different than single layer gels of the same formulation. The encapsulation of cells and the addition of phenol red within the hydrogel layers decreased bending modulus of the trilayer scaffolds. The data presented demonstrates that this fabrication method can make quasilaminates with robust interfaces, integrating layers of different mechanical properties and biofunctionalization, and thus forming the foundation for a multilaminate scaffold that more accurately represents native tissue.
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    Integrating valve-inspired design features into poly(ethylene glycol) hydrogel scaffolds for heart valve tissue engineering
    (Elsevier, 2015) Zhang, Xing; Xu, Bin; Puperi, Daniel S.; Yonezawa, Aline L.; Wu, Yan; Tseng, Hubert; Cuchiara, Maude L.; West, Jennifer L.; Grande-Allen, K. Jane
    The development of advanced scaffolds that recapitulate the anisotropic mechanical behavior and biological functions of the extracellular matrix in leaflets would be transformative for heart valve tissue engineering. In this study, anisotropic mechanical properties were established in poly(ethylene glycol) (PEG) hydrogels by crosslinking stripes of 3.4 kDa PEG diacrylate (PEGDA) within 20 kDa PEGDA base hydrogels using a photolithographic patterning method. Varying the stripe width and spacing resulted in a tensile elastic modulus parallel to the stripes that was 4.1-6.8 times greater than that in the perpendicular direction, comparable to the degree of anisotropy between the circumferential and radial orientations in native valve leaflets. Biomimetic PEG-peptide hydrogels were prepared by tethering the cell-adhesive peptide RGDS and incorporating the collagenase-degradable peptide PQ (GGGPQG↓IWGQGK) into the polymer network. The specific amounts of RGDS and PEG-PQ within the resulting hydrogels influenced the elongation, de novo extracellular matrix deposition and hydrogel degradation behavior of encapsulated valvular interstitial cells (VICs). In addition, the morphology and activation of VICs grown atop PEG hydrogels could be modulated by controlling the concentration or micro-patterning profile of PEG-RGDS. These results are promising for the fabrication of PEG-based hydrogels using anatomically and biologically inspired scaffold design features for heart valve tissue engineering.
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    Laminin Peptide-Immobilized Hydrogels Modulate Valve Endothelial Cell Hemostatic Regulation
    (Public Library of Science, 2015) Balaoing, Liezl Rae; Post, Allison Davis; Lin, Adam Yuh; Tseng, Hubert; Moake, Joel L.; Grande-Allen, K. Jane
    Valve endothelial cells (VEC) have unique phenotypic responses relative to other types of vascular endothelial cells and have highly sensitive hemostatic functions affected by changes in valve tissues. Furthermore, effects of environmental factors on VEC hemostatic function has not been characterized. This work used a poly(ethylene glycol) diacrylate (PEGDA) hydrogel platform to evaluate the effects of substrate stiffness and cell adhesive ligands on VEC phenotype and expression of hemostatic genes. Hydrogels of molecular weights (MWs) 3.4, 8, and 20 kDa were polymerized into platforms of different rigidities and thiol-modified cell adhesive peptides were covalently bound to acrylate groups on the hydrogel surfaces. The peptide RKRLQVQLSIRT (RKR) is a syndecan-1 binding ligand derived from laminin, a trimeric protein and a basement membrane matrix component. Conversely, RGDS is an integrin binding peptide found in many extracellular matrix (ECM) proteins including fibronectin, fibrinogen, and von Willebrand factor (VWF). VECs adhered to and formed a stable monolayer on all RKR-coated hydrogel-MW combinations. RGDS-coated platforms supported VEC adhesion and growth on RGDS-3.4 kDa and RGDS-8 kDa hydrogels. VECs cultured on the softer RKR-8 kDa and RKR-20 kDa hydrogel platforms had significantly higher gene expression for all anti-thrombotic (ADAMTS-13, tissue factor pathway inhibitor, and tissue plasminogen activator) and thrombotic (VWF, tissue factor, and P-selectin) proteins than VECs cultured on RGDS-coated hydrogels and tissue culture polystyrene controls. Stimulated VECs promoted greater platelet adhesion than non-stimulated VECs on their respective culture condition; yet stimulated VECs on RGDS-3.4 kDa gels were not as responsive to stimulation relative to the RKR-gel groups. Thus, the syndecan binding, laminin-derived peptide promoted stable VEC adhesion on the softer hydrogels and maintained VEC phenotype and natural hemostatic function. In conclusion, utilization of non-integrin adhesive peptide sequences derived from basement membrane ECM may recapitulate balanced VEC function and may benefit endothelialization of valve implants.
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    The characterization of the microstructure of the aortic valve for tissue engineering applications
    (2013-09-16) Tseng, Hubert; Grande-Allen, K. Jane; West, Jennifer L.; Jacot, Jeffrey G.; Killian, Thomas C.
    The aortic valve maintains unidirectional blood flow between the left ventricle and the systemic circulation. When diseased, the valve is replaced either by a mechanical or a bioprosthetic heart valve, that carry issues such as thrombogenesis, long term structural failure, and calcification, necessitating the development of more structurally and biologically sufficient long-term replacements. Tissue engineering provides a possible avenue for development, combining cells, scaffolds, and biochemical factors to regenerate tissue. The overall goal of this dissertation was to create a foundation for the rational design of a tissue engineered aortic valve. The novel approach taken in this thesis research was to view each of the three leaflets as a laminate structure. The first three aims consider the leaflet as a laminate structure comprising of layers of collagen, elastin, and glycosaminoglycans (GAGs). In the first aim, the effect of GAGs on the tensile properties and stress relaxation in the leaflet was investigated, by removing GAGs through increasing amounts of hyaluronidase. A decrease in GAGs led to significantly higher elastic moduli, maximum stresses, and hysteresis in the leaflet. In the second aim, the 3D elastic fiber network of the leaflet was characterized using immunohistochemistry and scanning electron microscopy. This structure was found to have regionally varying thicknesses and patterns. In the third aim, a novel hydrogel-fiber composite design was proposed to match the anisotropy of the leaflet. This composite composed of aligned electrospun poly(ε-caprolactone) (PCL) within a poly(ethylene glycol) diacrylate (PEGDA) matrix. Surface modification and embedding of the PCL did not significantly alter the anisotropy or strength of the underlying PCL scaffold, providing the basis for an anisotropic, biocompatible scaffold. In the last aim, a novel co-culture model was designed using magnetic levitation as a layered structure of valvular endothelial cells and interstitial cells. This technique was used to create co-culture models within hours, while maintaining cell phenotype and function, and inducing extracellular matrix formation, as shown by immunohistochemical stains and their gene expression profiling. The overall result of this dissertation is a clearer understanding of the layered structure-function relationship of the aortic valve, and its application towards heart valve tissue engineering.