Simple approach to optimize collagenase enzyme mixtures to improve cell recovery

Simple approach to optimize collagenase enzyme mixtures to improve cell recovery

Current state of the art

No simple method exists for defining optimal collagenase enzyme formulations for the recovery of primary cells from tissue. The unknown biochemical composition of traditional crude or enriched collagenase products, combined with variability in enzyme activities between each lot of the same product, precludes their use for this application.

One option is to use purified enzymes responsible for releasing cells from tissue: collagenase and neutral protease. However, their use is prohibited by the cost and expertise required to perform experiments that will provide meaningful data. Ideally, if you consider pursuing this path you need to engage an expert who is familiar with using design of experiment (DOE) methodologies and set enzyme activities that you believe will affect cell recovery. This may be difficult if effective enzyme activity ranges are unknown. Typically, a “screening DOE” must be performed and completed to determine the enzyme activity ranges to use in the critical DOE.

DE Collagenase Optimization kit: a fresh approach to the problem

The DE Collagenase optimization kit overcomes these limitations by aligning the design of the kit with fundamental principles of enzyme-mediated release of cells from the extracellular matrix (ECM). This model, summarized in an earlier blog post, is based on 6 core principles:

  1. The ECM is a collection of molecules secreted by cells into intercellular spaces, with the predominant matrix element being collagen fibrils or fibers.
  2. The ECM is resistant to degradation by proteases alone, but is readily degraded when protease is combined with functional collagenase enzymes that cuts collagen’s triple helical structure.
  3. Clostridium histolyticum collagenase[1] has very narrow specificity, only degrading native and denatured collagen, as measured by collagen degrading activity (CDA) and gelatinase activity, respectively
  4. Collagenase and neutral protease synergistically degrade the ECM:
    • Collagenase cuts native collagen’s triple helix native collagen that leads to the unraveled strands that are further degraded by neutral protease(s) and collagenase’s gelatinase activity.
    • Collagen degradation “loosens” the ECM, increasing the susceptibility of the ECM proteins to proteolysis.
  5. Once a sufficient number of the cell anchoring proteins in the ECM are degraded by proteases, the cells are released from tissue.
  6. If collagenase degrading activity (CDA) is in excess, the choice and dose of protease used in defined enzyme mixtures is the primary determinant for success of the cell isolation procedure.

By design, the 5 defined, enriched (DE) collagenase products are prepared by combining increasing amounts of enriched collagenase to a fixed amount purified BP Protease. Greater than 85% of the protein in the enriched collagenase preparation is intact C. histolyticum collagenase with high CDA. Purity of BP Protease (a Dispaseä equivalent enzyme) is > 95%. Tight control of both key enzyme activities enables a direct approach to determine the optimal enzyme composition and dose for cell recovery.

The key steps for optimizing the collagenase mass and protease activities via application of the DE Collagenase Optimization Kit are summarized below.

Step 1: Pre-requisites

  • A defined protocol for cell isolation, where you have expectations regarding cell yield, cell viability, or cell function.
  • You are currently using a collagenase-containing product, where the dose used for cell isolation is defined.
    • This “reference product” will be used to estimate the mass of collagenase to use in the experiments below.
    • If reference product is unavailable, the kit makes a recommendation of collagenase concentration for initial use.

Step 2, Experiment 1: Determine an acceptable protease activity for use in cell isolation procedure

  • The reference product provides an estimate of the collagenase mass used in the isolation procedure; calculations are performed to keep this mass constant when using the DE Collagenase 10, 20, 40, 60, and 80 products in the optimization experiment.
  • Since each DE product contains increasing amounts of collagenase, fixing this amount will result in decreasing the amount of protease used in the isolation procedure.
  • After plotting the results from 5 isolations using the products in the Optimization kit, 3 of the 4 potential patterns are shown in the graphs below where cell yield (or other readout measure) is plotted on the ordinate (y axis) and the neutral protease activity is plotted on the abscissa (x axis).
    • These plots are defined by the shape of the curve as sigmoidal, positive slope, or negative slope (the 4th type: “no slope”, where plotted results are a flat line, is not shown).
  • The peak of a sigmoidal curve identifies the optimal dose of NPA to use in the isolation procedure.
    • The other two curve types indicate that cell yield will increase if protease is added (positive slope) or reduced (negative slope).
    • In these two cases, further experiments will need to be performed if you want to improve NPA optimization in the isolation procedure
  • For purpose of illustration, assume that the sigmoidal curve was generated when plotting the results from this experiment.

Step 3, Experiment 2: Determine an acceptable collagenase activity for use in cell isolation procedure

  • The same procedure is used as described above, but in this case the NPA determined in Experiment 1 is held constant and the mass of collagenase is increased.
  • As above, after performing the 5 isolations, four possible curves are generated after plotting the data with the ideal result showing a sigmoidal curve. The collagenase mass at the peak of the curve is the optimal mass for the fixed NPA.
    • It is possible that the result may look more like a flat curve or a curve where cell yield increases, then plateaus.
    • This is not unexpected, since adding excess collagenase has minimal adverse effect on cell isolation.
  • The conclusion for experiments 1 & 2 is a defined enzyme composition, where an optimal dose will be determined in Experiment 3.

Step 4, Experiment 3: Determined the optimal dose of the defined enzyme mixture defined from results of Experiments 1 & 2

  • The determination of an optimal dose is similar to what you are currently doing in qualifying a new lot of traditional crude or enriched collagenase product.
  • The major difference is that now you know the enzyme composition of the enzyme mixture used to isolate cells.
  • The dose chosen should be on the plateau of the dose response curve, so minor errors in preparation of the enzyme solution will not adversely affect cell yield or any other measured parameter.

Benefits from approach

There are several benefits for optimizing enzyme compositions using the approach described above:

  1. The results from these experiments can be readily translated into development of a custom, GMP grade product, supporting validation of an enzyme composition and dose for use in clinical isolation procedures
  2. The breadth of collagenase:protease ratios is wider than what has been used in the past, enabling investigators to reduce the amount of enzyme required for cell isolation to levels lower than that expected with traditional collagenase products.
  3. If the results from experiments 1 or 2 do not generate a sigmoidal curve, VitaCyte can provide additional guidance on extending the range of the protease activity or collagenase mass used in the optimization experiments.
  4. The same principles outlined above can be applied to optimizing an collagenase enzyme mixture to isolate adherent cells after in vitro

Additional information

A detailed description of the method summarized below can be downloaded from the VitaCyte website in the white paper “DE Collagenase Optimization Kit: a fresh approach to defining enzyme composition and dose for maximal cell recovery”. If you have any questions on this method, call Technical Support at 317-917-3457, option 2 or send an e mail to feedback@vitacyte.com.

[1] C. histolyticum collagenase has a broad specificity to degrade all forms of mammalian native collagen in contrast to mammalian collagenases that have a narrow specificity as enzymes used in tissue remodeling

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