Pancreatic enzyme requirements for the dissociation of rat hearts for culture

Summary Nine crude commercially available samples of pancreatic enzyme preparations were examined in an effort to establish enzyme requirements for dissociation of rat heart to single cells for culture. Disc gel electrophoresis, using purified enzymes as references, indicated the presence of at least five enzymes. The levels of these enzymes, trypsin, chymotrypsin, elastase, lipase, and amylase were quantified in the commercial samples by established enzyme assays. The crude enzyme preparations were compared for their abilities to provide good yields of viable cells from the hearts of neonatal white rats. The abilities of the cells to thrive in culture and to beat rhythmically were also used in the comparison. Commercial purified samples of the five enzymes were used singly and in various combinations in preparing cultures to establish minimal pancreatic enzyme requirements for dissociation of rat hearts. It was concluded that at least trypsin, chymotrypsin, and elastase were required to obtain viable rat heart cells in high yield. Amylase and lipase activities were shown not to be necessary for dissociation. Most commercial samples of trypsin, commonly used to dissociate heart tissue, contain trypsin, chymotrypsin, and elastase in concentrations sufficient to release heart cells in good yield with minimal damage. The destruction of cells observed with some of the commercial samples examined was not due to improper levels of trypsin, chymotrypsin, or elastase. Work is in progress to identify the destructive agent(s). This work was supported in part by United States Public Health Service Grant HL10018 and The Pennsylvania State University Agricultural Experiment Station Grant 1858. Authorized for publication on December 28, 1973 as paper 4602 in the journal series of the Pennsylvania Agricultural Experiment Station.