In vitro evaluation of human muscle satellite cell migration prior to fusion into myotubes.
We developed a short-time assay to evaluate muscle satellite cell migration, based on the fact that during myogenic differentiation, myoblasts migrate preferentially towards high cellular density areas where myotubes would form. This assay consists of a computer-assisted count of cells within a randomly chosen field, performed every hour for eight hours, and compared with the cell number at the start time of the experiment. Nine primary myoblast cultures were tested in triplicate. The method relies on several requisites. (1) Negligible cell proliferation: cell division was nearly absent in 8 h experiments. (2) Directional cell movement: a major flow of cells, either entering or exiting the fields, was constantly observed. 'Counter-flows', detected by visual counting, involved minor percentages of cells. (3) Constant migration rate: a linear increase in cell count variations over 8 h and a very high degree of intra-assay homogeneity were observed. Individual primary cell culture characteristics (depending on characteristics of the different donors) were the sole factor with a significant impact on migration rate. Automatic cell counting conveniently assessed the inhibitory effect of GRGDTP, an inhibitor of integrin-mediated cell adhesion. The method described here is rapid, does not require heavy equipment, and allows studies under serum-free conditions required to test molecules interfering with cell migration, in the course of the in vitro myogenic process.
Chazaud
B
B
GERMEN (Groupe d'Etudes et de Recherches sur le Muscle et le Nerf) EA 2347-Université Paris XII Val-de-Marne, Créteil, France. chazaud@univ-paris12.fr
Christov
C
C
Gherardi
R K
RK
Barlovatz-Meimon
G
G
D002452:Cell Count / Q000379:methods; D002454:Cell Differentiation / Q000502:physiology; D002455:Cell Division / Q000502:physiology; D002465:Cell Movement / Q000502:physiology*; D002478:Cells, Cultured; D016895:Culture Media, Serum-Free / Q000494:pharmacology; D006801:Humans; D016023:Integrins / Q000502:physiology; D018485:Muscle Fibers, Skeletal / Q000737:chemistry / Q000166:cytology* / Q000187:drug effects; D018482:Muscle, Skeletal / Q000166:cytology / Q000196:embryology; D009842:Oligopeptides / Q000494:pharmacology
D016895:Culture Media, Serum-Free; D016023:Integrins; D009842:Oligopeptides; C080493:fibronectin attachment peptide