ABSTRACT
In this study, 3D culture system for human adipose-derived stem cell (hASC) using a BioLevitator as the bioreactor for microcarrier-based cultures was established. During the culturing period, hASCs preferred to grow in crevices between microcarriers and a high viability was maintained even when reaching confluency. Adipogenic or osteogenic differential medium was used to induce hASCs and differential potentials of these cells were compared between 2D and 3D environments via RT-PCR and staining quantifications. CEBP/α gene expression was significant higher in 3D condition at day 21 (P < 0.05). Staining quantification indicates that cells cultured in 3D condition have significant better differentiation potential from day 14 to 21 for both adipogenic and osteogenic lineages (P < 0.01).
Subject(s)
Cell Culture Techniques/methods , Cell Differentiation , Stem Cells/physiology , Adipose Tissue/cytology , Bioreactors , Cell Survival , Humans , Stem Cells/cytologyABSTRACT
Chemical vapor deposition polymerization was used to deposit a novel maleimide-functionalized poly-p-xylylene coating on various substrates. The coated substrates are readily able to undergo thiol-maleimide click reaction under mild conditions. Applications using this coating technology are highlighted in low-protein-fouling modification as well as manipulated attachments and growth of bovine arterial endothelial cells.
Subject(s)
Maleimides/chemistry , Maleimides/chemical synthesis , Piperidines/chemical synthesis , Xylenes/chemistry , Bioengineering , Click Chemistry , Piperidines/chemistry , Polymerization , Surface Properties , Volatilization , Xylenes/chemical synthesisABSTRACT
A new approach is presented to control cell attachment behavior on biocompatible substrates. Multiple layers of polylactic acid (PLA) were inkjet-printed on dry alginate films to create composite surfaces with rigidity variation. The printed films were submerged in cell culture medium and fibroblast 3T3-L1 cells were cultured on the printed films. 3T3-L1 cells were found to preferentially adhere on PLA surfaces with higher rigidity. The same approach was also used to create various cell attachment patterns. This study provides a new methodology to fabricate biodegradable matrix for favorable cell adhesion or patterning.
Subject(s)
Coated Materials, Biocompatible/pharmacology , Hardness/physiology , Lactic Acid/pharmacology , Polymers/pharmacology , Tissue Scaffolds/chemistry , 3T3-L1 Cells , Alginates/chemistry , Alginates/pharmacology , Animals , Cell Adhesion/drug effects , Cell Culture Techniques/instrumentation , Coated Materials, Biocompatible/chemistry , Glucuronic Acid/chemistry , Glucuronic Acid/pharmacology , Hexuronic Acids/chemistry , Hexuronic Acids/pharmacology , Lactic Acid/chemistry , Mice , Polyesters , Polymers/chemistry , Surface PropertiesABSTRACT
Reactive polymer coatings were synthesized via chemical vapor deposition (CVD) polymerization process. These coatings decouple surface design from bulk properties of underlying materials and provide a facile and general route to support thiol-ene and thiol-yne reactions on a variety of substrate materials. Through the reported technique, surface functions can be activated through a simple design of thiol-terminated molecules such as polyethylene glycols (PEGs) or peptides (GRGDYC), and the according biological functions were demonstrated in controlled and low-fouling protein adsorptions as well as accurately manipulated cell attachments.
Subject(s)
Polymers/chemistry , Sulfhydryl Compounds/chemistry , Alkenes/chemistry , Alkynes/chemistry , Amino Acid Sequence , Click Chemistry , Fibrinogen/chemistry , Gases/chemistry , Polymerization , Quinolinium Compounds/chemistry , Surface PropertiesABSTRACT
Upon cell adhesion to extracellular matrix proteins, focal adhesion kinase (FAK) rapidly undergoes autophosphorylation on its Tyr-397 which consequently serves as a binding site for the Src homology 2 domains of the Src family protein kinases and several other intracellular signaling molecules. In this study, we have attempted to examine the effect of the FAK Y397F mutant on v-Src-stimulated cell transformation by establishing an inducible expression of the Y397F mutant in v-Src-transformed FAK-null (FAK(-/-)) mouse embryo fibroblasts. We found that the FAK Y397F mutant had both positive and negative effects on v-Src-stimulated cell transformation; it promoted v-Src-stimulated invasion, but on the other hand it inhibited the v-Src-stimulated anchorage-independent cell growth in vitro and tumor formation in vivo . The positive effect of the Y397F mutant on v-Src-stimulated invasion was correlated with an increased expression of matrix metalloproteinase-2, both of which were inhibited by the specific phosphatidylinositol 3-kinase inhibitor wortmannin or a dominant negative mutant of AKT, suggesting a critical role for the phosphatidylinositol 3-kinase/AKT pathway in both events. However, the expression of the Y397F mutant rendered v-Src-transformed FAK(-/-) cells susceptible to anoikis, correlated with suppression on v-Src-stimulated activation of ERK and AKT. In addition, under anoikis stress, the induction of the Y397F mutant in v-Src-transformed FAK(-/-) cells selectively led to a decrease in the level of p130(Cas), but not other focal adhesion proteins such as talin, vinculin, and paxillin. These results suggest that FAK may increase the susceptibility of v-Src-transformed cells to anoikis by modulating the level of p130(Cas).
Subject(s)
Focal Adhesion Protein-Tyrosine Kinases/genetics , Mutation , Oncogene Protein pp60(v-src)/metabolism , Actins/chemistry , Agar/chemistry , Androstadienes/pharmacology , Animals , Anoikis , Binding Sites , Cell Adhesion , Cell Cycle , Cell Line , Cell Movement , Cell Transformation, Neoplastic , Collagen/chemistry , Crk-Associated Substrate Protein/metabolism , Drug Combinations , Enzyme Activation , Enzyme Inhibitors/pharmacology , Fibroblasts/metabolism , Focal Adhesions , Gelatin/chemistry , Genes, Dominant , Immunoblotting , Immunoprecipitation , Laminin/chemistry , Matrix Metalloproteinase 2/biosynthesis , Mice , Mice, Nude , Mice, Transgenic , Microscopy, Fluorescence , Neoplasm Invasiveness , Paxillin/chemistry , Phosphatidylinositol 3-Kinases/metabolism , Phosphorylation , Proteoglycans/chemistry , Proto-Oncogene Proteins c-akt/metabolism , Signal Transduction , Talin/chemistry , Time Factors , Transfection , Tyrosine/chemistry , Vinculin/chemistry , Wortmannin , src-Family Kinases/metabolismABSTRACT
Crk-associated substrate (Cas) is highly phosphorylated by v-Src and plays a critical role in v-Src-induced cell transformation. In this study, we found that the Src homology (SH) 3 domain of Cas blocked v-Src-stimulated anchorage-independent cell growth, Matrigel invasion, and tumor growth in nude mice. Biochemical analysis revealed that the Cas SH3 domain selectively inhibited v-Src-stimulated activations of AKT and JNK, but not ERK and STAT3. Attenuation of the AKT pathway by the Cas SH3 domain rendered v-Src-transformed cells susceptible to apoptosis. Inhibition of the JNK pathway by the Cas SH3 domain led to suppression of v-Src-stimulated invasion. Taken together, our results indicate that the Cas SH3 domain has an anti-tumor function, which severely impairs the transforming potential of v-Src.
