A common procedure used to isolate primary cells is to dissociate the tissue with a bacterial enzymatic mixture containing collagenase. The most commonly used reagent is “crude” collagenase, an enzyme mixture minimally purified from Clostridium histolyticum culture supernatants that contains collagenase and other proteases¹. The lot to lot variability in the enzyme composition and performance in the customer’s application directly reflects the biological variability of the culture conditions. Most customers “pre-qualify” lots by testing a sample from a specific lot in their cell isolation application prior to purchase of additional product. This post discusses a method to reveal the collagenase profile found in lots of crude collagenase used successfully in cell isolation procedures with the goal of developing protocols for these applications using purified collagenases.

Crude Collagenase

Typically, crude collagenase is a brown to black lyophilized powder that contains many different fermentation by-products and endotoxin. The dark color reflects the pigmented proteins that are a byproduct generated during the bacterial fermentation process. In contrast, “enriched” collagenase products are lighter colored since additional steps are taken to reduce the level of pigmented protein and increase the concentration of the collagenase and other proteolytic enzymes present in the product. Many earlier reports have shown that enzyme mixtures containing purified collagenase and neutral protease gave results similar or superior to those obtained with crude collagenase products^2,3. In previous posts we have discussed the importance of using the correct assay for determining collagenase and neutral protease activity. The ability to correlate the different molecular forms of collagenase by HPLC analysis and collagen degrading activity (CDA) provides a unique insight into the collagenase enzyme compositions that are required for cell isolation procedures.

As noted in an earlier post, analysis of enriched or purified collagenase preparations by analytical anion-exchange HPLC can resolve Class II (C2) collagenase from Class I (C1) collagenase. Our analysis supported by earlier studies⁴ shows that there are 3 different molecular forms of C1: intact or full-length C1 with two collagen binding domains (CBD) and two truncated forms (C1b, C1c), each having a single collagen binding domain with the C terminal CBD lost due to proteolysis during the fermentation or culture process⁵. In contrast, HPLC analysis of a crude collagenase mixture on the same analytical anion-exchange column does not provide useful information since the undefined pigments, which account for a majority of crude collagenase mixtures, obscures the collagenase peaks. Figure 1 below shows an HPLC profile of a crude product in which individual collagenase forms are indistinguishable. If one were to apply more crude mixture to the column irreparable damage of the resin can occur since the pigment often precipitates and binds irreversibly to the column.

Figure 1

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Pigment Removal

The analysis of crude collagenase by HPLC is improved if the pigment is reduced by passage over an affinity resin. This procedure allows for identification and quantitation of the various molecular forms of collagenase (see Figure 2 below after pigment removal). HPLC analysis of these enriched preparations show that this lot of crude collagenase contains primarily intact C1 with relatively little amounts of either of the degraded C1 molecular forms. Similar analyses of crude collagenases from other vendors showed that most lots have a highly variable amount of C2 (20-40%) when expressed as the total amount of collagenase by area under the curve. In addition, the amount of intact and degraded C1 varies dependent upon the vendor and lot of the product.

Figure 2

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The analysis summarized above indicates how much contaminating non-enzymatic protein and pigment is present in crude collagenase preparations. When three lots of crude collagenases were analyzed by the FITC-labeled collagen fibrils, the specific CDA ranged from 2200 to 5100 U/mg. However, after pigment removal the specific CDA of these collagenase mixtures were 28,325 U/mg and 35,391 U/mg for the two samples with minimal degraded C1 and 9,632 U/mg for the sample with a significant contribution of degraded C1 (Figure 3). Using this approach we estimated that in certain crude collagenase TDE products as little as 4% of the product is active enzyme.

Figure 3

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Conclusion

Biochemical analysis after affinity resin pigment removal of crude collagenase preparations used successfully for cell isolation provided insight into the collagenase component required for TDE for a particular tissue type. A key component of this analysis is the integration of the CDA with the analytical HPLC analysis to assess the different molecular forms of collagenase required for successful cell isolation. This approach has been used by our laboratory to prepare a defined TDE product for isolation of human hepatocytes (reference earlier post). We believe that this analysis is applicable to development of specific purified enzyme compositions used in the isolation of any primary cell population.

References

1.   Peterkofsky B. (1982) Bacterial Collagenase. Methods in Enzymology 82, 453-471.

2.   Hefley T.J., Stern P.H., and Brand J.S. (1983) Enzymatic isolation of cells from neonatal calvaria using two purified enzymes from Clostridium histolyticum. Experimental Cell Research 149, 227-236.

3.   Linetsky E., Bottino R., Lehmann R., Alejandro R., Inverardi L., and Ricordi C. (1997) Improved human islet isolation using a new enzyme blend, liberase. Diabetes 46, 1120-1123.

4.   Hefley T.J. (1987) Utilization of FPLC-purified bacterial collagenase for the isolation of cells from bone. J Bone and Mineral Research 2, 505-516.

5.   McCarthy R.C., Spurlin B., Wright M.J., Breite A.G., Sturdevant L.K., Dwulet C.S., and Dwulet F.E. (2008) Development and characterization of a collagen degradation assay to assess purified collagenase used in islet isolation. Transplantation Proceedings 40[2], 339-342.

Categories: Cell Isolation, Collagenase, Tissue Dissociation