Fabrication of oxygen-carrying microparticles functionalized with liver ECM-proteins to improve phenotypic three-dimensional in vitro liver assembly,function, and responses

Oxygen and extracellular matrix (ECM)-derived biopolymers play vital roles in regulating many cellular functions in both the healthy and diseased liver. This study highlights the significance of synergistically tuning the internal microenvironment of three-dimensional (3D) cell aggregates composed of hepatocyte-like cells from the HepG2 human hepatocellular carcinoma cell line and hepatic stellate cells (HSCs) from the LX-2 cell line to enhance oxygen availability and phenotypic ECM ligand presentation for promoting the native metabolic functions of the human liver. First, fluorinated (PFC) chitosan microparticles (MPs) were generated with a microfluidic chip, then their oxygen transport properties were studied using a custom ruthenium-based oxygen sensing approach. Next, to allow for integrin engagements the surfaces of these MPs were functionalized using liver ECM proteins including fibronectin, laminin-111, laminin-511, and laminin-521, then they were used to assemble composite spheriods along with HepG2 cells and HSCs. After in vitro culture, liver-specific functions and cell adhesion patterns were compared between groups and cells showed enhanced liver phenotypic responses to laminin-511 and 521 as evidenced via enhanced E-cadherin and vinculin expression, as well as albumin and urea secretion. Furthermore, hepatocytes and HSCs exhibited more pronounced phenotypic arrangements when cocultured with laminin-511 and 521 modified MPs providing clear evidence that specific ECM proteins have distinctive roles in the phenotypic regulation of liver cells in engineering 3D spheroids. This study advances efforts to create more physiologically relevant organ models allowing for well-defined conditions and phenotypic cell signaling which together improve the relevance of 3D spheroid and organoid models.     

Inhibition of Collagen Fibrillogenesis by Cells Expressing Soluble Extracellular Domains of DDR1 and DDR2

Collagen fiber assembly affects many physiological processes and is tightly controlled by collagen-binding proteins. However, to what extent membrane-bound versus cell-secreted collagen-binding proteins affect collagen fibrillogenesis is not well understood. In our previous studies, we had demonstrated that the membrane-anchored extracellular domain (ECD) of the collagen receptor discoidin domain receptor 2 (DDR2) inhibits fibrillogenesis of collagen endogenously secreted by the cells. These results led to a novel functional role of the DDR2 ECD. However, since soluble forms of DDR1 and DDR2 containing its ECD are known to naturally exist in the extracellular matrix, in this work we investigated if these soluble DDR ECDs may have a functional role in modulating collagen fibrillogenesis. For this purpose, we created mouse osteoblast cell lines stably secreting DDR1 or DDR2 ECD as soluble proteins. Transmission electron microscopy, fluorescence microscopy, and hydroxyproline assays were used to demonstrate that DDR ECD expression reduced the rate and quantity of collagen deposition and induced significant changes in fiber morphology and matrix mineralization. Collectively, our studies advance our understanding of DDR receptors as powerful regulators of collagen deposition in the ECM and elucidate their multifaceted role in ECM remodeling.     

Extracellular matrix proteins regulate epithelial–mesenchymal transition in mammary epithelial cells

Mouse mammary epithelial cells undergo transdifferentiation via epithelial–mesenchymal transition (EMT) upon treatment with matrix metalloproteinase-3 (MMP3). In rigid microenvironments, MMP3 upregulates expression of Rac1b, which translocates to the cell membrane to promote induction of reactive oxygen species and EMT. Here we examine the role of the extracellular matrix (ECM) in this process. Our data show that the basement membrane protein laminin suppresses the EMT response in MMP3-treated cells, whereas fibronectin promotes EMT. These ECM proteins regulate EMT via interactions with their specific integrin receptors. α6-integrin sequesters Rac1b from the membrane and is required for inhibition of EMT by laminin. In contrast, α5-integrin maintains Rac1b at the membrane and is required for the promotion of EMT by fibronectin. Understanding the regulatory role of the ECM will provide insight into mechanisms underlying normal and pathological development of the mammary gland.     

Dynamic remodeling of the extra cellular matrix during zebrafish fin regeneration

Extracellular matrix plays a dynamic role during the process of wound healing, embryogenesis and tissue regeneration. Caudal fin regeneration in zebrafish is an excellent model to study tissue and skeletal regeneration. We have analyzed the expression pattern of some of the well characterized ECM proteins during the process of caudal fin regeneration in zebrafish. Our results show that a transitional matrix analogous to the one formed during newt skeletal and heart muscle regeneration is synthesized during fin regeneration. Here we demonstrate that a provisional matrix rich in hyaluronic acid, tenascin C, and fibronectin is synthesized following amputation. Additionally, we observed that the link protein Hapln1a dependent ECM, consisting of Hapln1a, hyaluronan and proteoglycan aggrecan, is upregulated during fin regeneration. Laminin, the protein characteristic of differentiated tissues, showed only modest change in the expression pattern. Our findings on zebrafish fin regeneration implicates that changes in the extracellular milieu represent an evolutionarily conserved mechanism that proceeds during tissue regeneration, yet with distinct players depending on the type of tissue that is involved.     

Transport of Catecholamines by Native and Reconstituted Rat Heart Synaptic Vesicles

Highly purified “heavy” synaptic vesicles were isolated from rat heart by differential centrifugation. Because of the high intravesicular concentrations of proteins, catecholamine, and ATP, resealed vesicle ghosts were prepared and used to study the detailed kinetics of catecholamine transport. ATP stimulated the uptake of /-norepinephrine and was saturable with a K_m for l-norepinephrine at 3.3 μM and 1.8 mM for ATP. The ghosts also accumulated 5-hydroxytryptamine and l-epinephrine via an ATP-dependent mechanism. Uptake was stereospecific for the l-form. A functional catecholamine transporter could be solubilized by the detergent octyl-glucoside and incorporated into phospholipid vesicles, which, after detergent removal, generated proteoliposomes that accumulated l-norepinephrine. Reserpine- and l-propranolol-sensitive accumulation against a concentration gradient is achieved by artificially creating a pH gradient across the membrane, and lends further support to the idea that at least the initial phase of catecholamine transport is driven by the trans-membrane pH gradient created by the proton-translocating ATPase.     

Interommatidial cells build a tensile collagen network during Drosophila retinal morphogenesis

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In vitro lung cancer multicellular tumor spheroid formation using a microfluidic device

The purpose of this study was to demonstrate self-organizing in vitro multicellular tumor spheroid (MCTS) formation in a microfluidic system and to observe the behavior of MCTSs under controlled microenvironment. The employed microfluidic system was designed for simple and effective formation of MCTSs by generating nutrient and oxygen gradients. The MCTSs were composed of cancer cells, vascular endothelial cells, and type I collagen matrix to mimic the in vivo tumor microenvironment (TME). Cell culture medium was perfused to the microfluidic device loaded with MCTSs by a passive fluidic pump at a constant flow rate. The dose response to an MMPs inhibitor was investigated to demonstrate the effects of biochemical substances. The result of long-term stability of MCTSs revealed that continuous perfusion of cell culture medium is one of the major factors for the successful MCTS formation. A continuous flow of cell culture medium in the in vitro TME greatly affected both the proliferation of cancer cells in the micro-wells and the sustainability of the endothelial cell-layer integrity in the lumen of microfluidic channels. Addition of MMP inhibitor to the cell culture medium improved the stability of the collagen matrix by preventing the detachment and shrinkage of the collagen matrix surrounding the MCTSs. In summary, the present constant flow assisted microfluidic system is highly advantageous for long-term observation of the MCTS generation, tumorous tissue formation process and drug responses. MCTS formation in a microfluidic system may serve as a potent tool for studying drug screening, tumorigenesis and metastasis.