Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone

Abstract

Crude bacterial collagenase is essential for the enzymatic isolation of cells from the membranous bone of neonatal mouse calvaria. We have employed the newly developed methodology of fast protein liquid chromatography (FPLC) to separate and quantify the isozymes of collagenase so that their role in the enzymatic isolation of cells might be determined. FPLC resolved as many as six protein peaks in less than 30 min using a single anion exchange column and separated collagenase isozymes into two classes. The Class I isozymes had a preference for the substrate Azocoll, a denatured collagen substrate, and the Class II isozymes had a preference for Hexapeptide, a synthetic substrate. Two preparations of chromatographically purified collagenase (CGN-A and CGN-B) were tested for their ability to release viable cells from bone. Both preparations of purified collagenase completely digested the calvaria in 120 min. The total cell yield obtained with CGN-A was 0.34 million cells per calvarium. The yield obtained with CGN-B was 1.01 million cells per calvarium. Each preparation of purified collagenase was analyzed using FPLC. CGN-A contained only Class I collagenase isozymes, whereas CGN-B contained a mixture of both Class I and Class II isozymes. The collagenase isozymes of CGN-B were separated by FPLC and then combined in a 4:1 ratio of Class II:Class I isozymes. Utilization of FPLC-separated collagenase isozymes for the cell isolation increased the total cell yield to 1.50 million cells/calvarium. We have concluded that there are many combinations of collagenase isozymes that will completely digest the extracellular matrix of bone. However, only a combination which favors the Class II isozymes will result in a low rate of cell destruction and high cell yields.