The optimal methodology of isolation and culturing myogenic satellite cells from adult rat skeletal muscle has been established during the last year. Rat satellite cells were purified from adult rat gastrocnemius hind-leg muscle. The muscle fragments were digested for 2 hours at 37o using collagenase and then plated in DMEM plating medium containing 1% antibiotics, 1% Glutamine, 10% Horse serum and 3.5% Hepes. After 3 days the plating medium was replaced with a proliferating medium containing all the above plus 20% FCS (Foetal Calf Serum). Passages and medium replacements have been performed until the flasks have reached a 90%-100% confluence. The cells were irradiated at this stage by Ga-As diode laser at an energy density of 4 J/cm2. BrdU at a concentration of 8μg/ml medium was added to the culture medium in order to label the nuclei of the dividing cells and enable their identification in the myocardium tissue sections after injection. This is done by immunostaining methods, as described later. Then the cells were harvested with Trypsin, counted and collected (after centrifugation) into 0.1 ml saline for injection.
One-1.5x106 cells were injected into 2-3 locations of the myocardium of the left ventricle of the beating heart. There was a high mortality rate (of about 60%) when using this procedure. The rats were then left as untreated Controls or laser irradiated (Ga-As laser 808nm) at a power density of 8mW/cm2 on the myocardium. The laser irradiation was performed immediately and 14 days post implantation. All rats were sacrificed at 30 days after implantation. The hearts were excised, sliced to 5 cross sections each of about 2mm thick and fixed in Neutral Buffered Formaldehyde. The tissue was processed for histological paraffin. Immunostaining for BrdU was performed using the Zymed 93-3943 BrdU staining Kit.
The identification of BrdU positive cells (injected cells) indicated groups of 2-10 cells at various locations in the left ventricle that represent the implanted cells. Sections from 2 control and 2 laser irradiated rats were analyzed so far. Other sections from 5 Controls and 5 laser irradiated rats are awaiting to be analyzed.
In summary, the methodology for isolation and labeling of satellite cells from skeletal muscles has been established as well as a reproducible method for injecting them into the beating myocardium. Analysis of the results for the possible better survival of the implanted cells by laser irradiation will be finalized in about 2 months.