Procedures for harvesting satelite cells from equine skeletal muscle

The objective of this study was to determine if equine muscle satellite cells (ESC) could be harvested, stored, and cultured in vitro using techniques established previously. Gluteus medius muscles of Quarter Horse yearlings, dissected free of connective and adipose tissue, were rinsed in phosphate buffered saline (PBS), and minced by hand. Minced tissue was weighed, transferred into sterile centrifuge tubes (approximately 30 g per tube), and incubated in pr0nase (1 mg/ml in PBS) at 37°C for 60 minutes to liberate satellite cells. Centrifuge tubes were agitated manually at 15-minute intervals to ensure adequate exposure to pronase. Following the incubation, cells were pelleted by differential centrifugation. Equine satellite cells were separated from fibroblasts by a 2-hour incubation in McCoys 5A medium (containing 10% horse serum) in uncoated 144 mm² culture dishes. The satellite cell suspension was drawn off and cells pelleted by centrifugation (1600 x g). Equine satellite cells were resuspended in McCoys 5A medium containing 20% fetal bovine serum and 10% dimethyl sulfoxide and frozen in liquid nitrogen in 3-ml aliquots. To ensure success in procedures to liberate ESC from muscle, thawed ESC were cultured in 24-well plates in McCoys 5A medium containing 10% horse serum and maintained in a humidified atmosphere of 95% 0₂ and 5% CO₂. After 24 hours, cultures were washed three times with McCoys medium containing 1% antibiotic antimycotic to remove cellular debris and dimethyl sulfoxide. Cells were monitored and fed every other day for the first week in culture. Thereafter, culture medium was changed less frequently to allow serum to become depleted and differentiation to occur. Equine satellite cells were able to proliferate, differentiate and form myotubes in culture. Thus, this methodology seems appropriate to study ESC.