Most lyophilized collagenase containing tissue dissociation enzymes (TDEs) used today have a wide spectrum of color – tan to black – depending on the product. This color serves as a litmus test indicating that these enzymes are not pure and are likely have high variability in enzyme activity between lots. The information below presents more details of the biochemical characteristics of crude or enriched collagenase products and shows the benefits of using purified enzymes in place of these products.
Commonly, crude or enriched collagenase products contain pigments (responsible for the color), endotoxin, undesirable proteases and other proteins and proteolytic fragments. Lot qualification is necessary to verify that the product performs acceptably in cell isolation with regards to cell yield, viability, and function. Analytical anion exchange high pressure chromatography analysis (HPLC) is an excellent method to further analyze the heterogeneity of these materials (for more detail on HPLC see the post from October 2009). The top HPLC chromatogram below is an analysis of a representative lot of crude collagenase used for ADSC isolation. This material contains primarily pigment with less than 4% of the protein in the sample reflecting collagenase. Analysis of several other lots of crude collagenase from Worthington or from Sigma has shown similar chromatograms.
Analytical Anion-Exchange HPLC of Crude Collagenase
To gain additional insight of the quality of collagenase, a small portion of this product was further purified at VitaCyte using an affinity chromatography procedure to reduce the pigment concentration and increase the proportion of collagenase in the sample. The bottom chromatogram is an analysis of the affinity purified, enriched collagenase. This chromatogram is consistent with similar analyses performed on the Sigma Type XI enriched collagenase product. It has a higher specific collagenase activity as measured by collagen degradation activity units (CDA U) per mg protein when compared to crude collagenase, indicating the degree of purification.
Analytical Anion-Exchange HPLC of Affinity Purified Crude Collagenase
How can you avoid this problem?
In contrast to the preparations described above, VitaCyte purifies each component to a single enzyme then sells the purified enzyme separately (i.e., neutral protease) or adds that component back into a purified and defined enzyme mixture (purified collagenase). This approach leads to a reproducibly manufactured, consistent material that contains very low amounts of the contaminants associated with crude or enriched products, e.g., extraneous enzyme activity, pigment, endotoxin. Purity equates consistent and improved performance in tissue dissociation. An unspoken concern for any scientist using a poorly characterized reagent is that the results obtained from the experiment may not be reproducible. The use of a purified enzymes removes this concern since the same enzyme composition is found in each and every lot of each product.
VitaCyte Purified Collagenase HPLC
(click to enlarge)
Eliminate the Noise and Gain Control
Benefits of using purified tissue dissociation enzymes from VitaCyte
- Lot to lot variation minimized
- Endotoxin reduced, contaminants eliminated
- Blends can be modified for further optimization
- Same enzyme mixture can be used by collaborating labs, increasing the level of productive work