Investigating conversion of mechanical force into biochemical signaling in three-dimensional chondrocyte cultures.
The culture of chondrocytes embedded within agarose hydrogels maintains chondrocytic phenotype over extended periods and allows analysis of the chondrocyte response to mechanical forces. The mechanisms involved in the transduction of a mechanical stimulus to a physiological process are not completely deciphered. We present protocols to prepare and characterize constructs of murine chondrocytes and agarose (1 week pre-culture period), to analyze the effect of compression on mRNA level by RT-PCR (2-3 d), gene transcription by gene reporter assay (3 d) and phosphorylation state of signaling molecules by western blotting (3-4 d). The protocols can be carried out with a limited number of mouse embryos or newborns and this point is particularly important regarding genetically modified mice.
Bougault
Carole
C
UMR5086, CNRS, IFR128, IBCP (Institut de Biologie et Chimie des Protéines), Université de Lyon, Lyon, France.
Paumier
Anne
A
Aubert-Foucher
Elisabeth
E
Mallein-Gerin
Frédéric
F
D000818:Animals; D000831:Animals, Newborn; D001483:Base Sequence; D001696:Biomechanical Phenomena; D015153:Blotting, Western; D018929:Cell Culture Techniques / Q000379:methods*; D002469:Cell Separation; D019902:Chondrocytes / Q000166:cytology / Q000502:physiology*; D024043:Collagen Type II / Q000235:genetics; D017931:DNA Primers / Q000235:genetics; D004622:Embryo, Mammalian / Q000166:cytology; D015870:Gene Expression; D017930:Genes, Reporter; D006801:Humans; D020100:Hydrogels; D020935:MAP Kinase Signaling System / Q000502:physiology; D040542:Mechanotransduction, Cellular / Q000502:physiology*; D051379:Mice; D010766:Phosphorylation; D011401:Promoter Regions, Genetic; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism; D020133:Reverse Transcriptase Polymerase Chain Reaction; D012685:Sepharose; D015398:Signal Transduction / Q000502:physiology*; D013314:Stress, Mechanical; D014162:Transfection
C499177:COL2A1 protein, human; D024043:Collagen Type II; D017931:DNA Primers; D020100:Hydrogels; D012333:RNA, Messenger; D012685:Sepharose