Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum

The enzymatic isolation of cells with bacterial collagenase has proved to be a powerful technique for the study of a wide variety of tissues. Unfortunately, for some applications such as the isolation of cells from membranous bone, the cellular damage that results from the exposure of the cells to cytotoxic contaminants of bacterial collagenase has limited the usefulness of this approach. The use of chromatographically purified collagenase alone is often ineffective or very slow to release cells from tissue. We have found that two enzymes are necessary and sufficient to isolate cells from neonatal mouse calvaria: purified collagenase and neutral protease. These two enzymes can be chromatographically purified on a preparative scale to yield 100 mg amounts of each enzyme. The purified enzymes can be recombined in amounts which will digest calvaria at the same rate as the crude bacterial collagenase from which they were derived. The cells that are isolated using the purified enzymes are undamaged, as indicated by the measurement of their equilibrium density on gradients of Ficoll and sodium metrizoate. Cells isolated with crude collagenase never reach an equilibrium density upon isopyknic centrifugation, whereas cells isolated with the purified enzymes reach an equilibrium density of 1.074 g/ml in 90 min.