Collagenase lot selection and purification for adipose tissue digestion

Crude Clostridial collagenase (CCC) remains the most widely used enzyme for the digestion of tissues prior to cell isolation and culture. CCC contains numerous components in addition to specific collagenases and proteases. A chronic problem associated with CCC is significant lot variability which occurs with respect to the ability of different lots of CCC to digest tissue. We have evaluated numerous commercially available samples of CCC for their ability to digest human liposuction-derived SC fat. Digestion capacity was evaluated as the ability to release endothelial cells from fat as well as the ability of isolated cells to adhere to tissue culture plastic. A significant variation in digestion efficacy between lots of collagenase was observed. We subsequently purified CCC using a partial purification method with dialysis and centrifugation as well as a complete purification, using liquid chromatography, to remove all nonspecific proteases. While partially purified collagenase retained digestion capacity, pure collagenase exhibited reduced digestion capacity. Maximum digestion was achieved with pure collagenase when trypsin was added. The use of completely purified collagenase with trypsin is advantageous where all components in the enzyme digestion mixture must be known.