Enzyme-mediated cell isolation methods require tissue dissociation enzymes (TDEs) that contain optimal amounts of C. histolyticum collagenase and neutral protease activity. Salt-precipitated C. histolyticum culture supernatants were first sold as lyophilized products in the 1960s. These traditional collagenase products required researchers to pre-qualify new lots in an application before purchase. In the 1990s, reverse engineering of “good lots” of collagenase for specific applications led to use of purified enzymes that improved product consistency but did not address TDE optimization.

A mechanistic model for collagenase-protease mediated cell isolation reported in 2011 proposed that collagenase only degrades collagen, which leads to thinning of the collagen jungle found in the extracellular matrix (ECM), that in turn leads to exposure of protease-sensitive sites on ECM cell-anchoring proteins. Once proteases cut these anchoring proteins, cells are released from tissue.

The RDMTDE method is derived from the above model. It simplifies optimization of TDEs to one variable: neutral protease activity. The method requires that excess collagenase activity must be present in an enzyme-mediated cell isolation method that consistently provides sufficient yields of functional, viable cells; and the maximal amount of neutral protease activity required for cell recovery is ≤ the activity in Worthington’s Type 1 Collagenase.

The method has two requirements. First, you must determine the collagenase used in your current method (Reference Method). Second, it is necessary to use purified and characterized collagenase and neutral proteases to define the enzyme activities required for cell isolation.

The 3 steps of the process are:
1. Estimate the mass of collagenase in the enzyme solution used in the Reference Method
2,  Prepare solutions of collagenase-protease mixtures where the mass of collagenase is fixed and the neutral protease activity tittered to assess its effectiveness to release cells from tissue. A TDE Excel Calculator simplifies preparation of these solutions by entering the mass of collagenase (same for all solutions), volume of enzyme solution, and the amount of neutral protease activity expressed as the percentage found in Worthington Collagenase Type 1. The output from the calculator defines the amount of PD Collagenase 800 and Collagenase Gold to prepare each solution.
3. Isolate cells using the above solutions and compare the results to those using your current collagenase product.

The results from these isolations should be comparable to the Reference Method. If not, purified clostripain can be added to improve cell yields.

The method's primary benefit is defining the TDE composition required for cell isolation. This method can be used by any user of traditional or purified collagenase products. Once the enzyme composition is defined, further improvement can be made to determine the effect of these modifications on functional cell yields.