The use of 3-D culture in peptide hydrogel for analysis of discoidin domain receptor 1-collagen interaction.
The aim of this study is to examine a novel drop culture model using a biologically inspired self-assembling peptide: hydrogel (RAD16-I, also called PuraMatrix), which produces a nanoscale environment similar to native extracellular matrix (ECM) for a cell line weakly adherent to a plastic surface during cell culture. Our work investigates quantitatively analyzing discoidin domain receptor (DDR) 1-mediated protein interactions between collagen type I and matrix metalloproteinase (MMP)-2 or -9, as well as cell invasion, using, as a scaffold, PuraMatrix, a novel peptide hydrogel. Results demonstrate that the dynamic cell culture technique produced a highly stable reharvesting of cells throughout the constructs with HP-75, human pituitary adenoma cell line when compared to the traditional seeding methods. Secretion of MMP via collagen type I was observed quantitatively in the supernatant (EC50; MMP-2, 50.4 ng/ml: MMP-9, 57.6 ng/ml). In PuraMatrix gel impregnated with 50 ng/ml of collagen type I, transfection of the vector encoding full-length DDR1 or siRNA targeting DDR1 up- or downregulated respectively secretion of MMP-2 and -9, and cell invasion. Our results show that incorporation of this peptide with each ECM component provides a more permissive environment to elucidate ECM to cell signal interaction.
Yoshida
Daizo
D
Department of Neurosurgery, Nippon Medical School, Nippon Medical School, Tokyo, Japan. dyoshida@nms.ac.jp
Teramoto
Akira
A
D018929:Cell Culture Techniques / Q000379:methods*; D045744:Cell Line, Tumor; D003094:Collagen / Q000378:metabolism*; D000070818:Discoidin Domain Receptors; D006801:Humans; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D020778:Matrix Metalloproteinase 2 / Q000378:metabolism; D020780:Matrix Metalloproteinase 9 / Q000378:metabolism; D008855:Microscopy, Electron, Scanning; D009361:Neoplasm Invasiveness / Q000473:pathology; D010455:Peptides; D011485:Protein Binding; D020794:Receptor Protein-Tyrosine Kinases / Q000378:metabolism*; D011975:Receptors, Mitogen / Q000378:metabolism*
D010455:Peptides; D011975:Receptors, Mitogen; D020136:Hydrogel, Polyethylene Glycol Dimethacrylate; D003094:Collagen; D000070818:Discoidin Domain Receptors; D020794:Receptor Protein-Tyrosine Kinases; D020778:Matrix Metalloproteinase 2; D020780:Matrix Metalloproteinase 9