IN-VITRO DIFFERENTIATION OF MYOBLASTS FROM SKELETAL-MUSCLE OF RAINBOW-TROUT

Substrata, plating densities and tissue culture media were compared fo r their effects on the proliferation and differentiation of myoblasts from skeletal muscle of rainbow trout. Mono-nuclear cells were isolate d from the lateralis muscle of 4-11-month-old trout and plated on to g lass coverslips coated with fibronectin, laminin or Matrigel. Cell pro liferation was estimated by determining the density of nuclei on succe ssive days in culture, and myoblast differentiation was detected by im munostaining cultures with the myosin-specific monoclonal antibody MF2 0. Mononuclear cell proliferation was highest for cells cultured on fi bronectin or laminin and lowest for cells cultured on Matrigel, but th e total number of nuclei in myosin-positive cells did not differ betwe en substrata. The percentage of nuclei in myosin-positive myocytes and myotubes was significantly higher for cells cultured on Matrigel. The proportion of cells adhering to Matrigel and undergoing differentiati on increased with plating density. Of three media tested, Dulbecco's M odified Eagle Medium (DMEM), RPMI 1640 (RPMI), Leibovitz's L-15 (L-15) supplemented with 1 or 10% fetal bovine serum (FBS), a significantly greater proportion of the myoblasts differentiated when cells were cul tured in L-15+10% FBS. These results suggest that culturing trout musc le-derived cells on a substratum of Matrigel at a high density and mai ntaining cells in L-15 + 10% FBS provide the conditions that maximize the proportion of cells that actively synthesize muscle myosin and fac ilitate trout myoblast differentiation in vitro.