This post is password protected. To view it please enter your password below:

Password:

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

(click to enlarge)

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

(click to enlarge)

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

Several combinations of collagenase and neutral proteases were evaluated at different concentrations using a protocol based on that of Gotoh in which the TDEs are perfused directly into the pancreatic duct⁵. In all cases, the purified enzymes were compared to the yield and morphology of islets obtained with Sigma Collagenase Type XI (Type XI). As with islets isolated from other species, differences were observed between thermolysin and other neutral proteases. Smaller islets, lower yields and greater loss of morphology were observed after culture when thermolysin was used in the enzyme mixture. In contrast, islets isolated with BP Protease (analogous to Dispase®) were of similar size and morphology to those obtained after isolation with Type XI. The collagenase activity also impacts islet yield and morphology as the CIzyme Collagenase MA was comparable to Type XI and superior to those isolated with CIzyme Collagenase HA with average yields of 140, 130 and 87 islets per organ, respectively from C57/B6 mice.

Representative density gradient purified mouse islets obtained using VitaCyte’s purified tissue dissociating enzymes. Image courtesy of Dr. Raghu Mirmira and Natalie Stull of the Wells Center for Pediatric Research, Basic Diabetes Research Group at Indiana University School of Medicine

Significant variability in average islet yields from different strains of rats and mice have been reported. The major advantages of using purified TDEs in this application are once the VitaCyte enzyme composition is defined, the lot to lot consistency is assured. Additional modifications of the composition can be made to improve the yield and quality of islets recovered from different mouse strains. The major benefit of using purified TDEs in this application is the elimination of the labor intensive process of lot qualification normally required for enriched collagenase products. Reporting optimal enzyme compositions for isolation of islets from different strains of mice can be confidently communicated, leading to more productive used of resources to address the needs of ongoing research programs.

Reference List

1.    Nikolova G, Jabs N, Konstantinova I, et al (2006) The vascular basement membrane: a niche for insulin gene expression and beta cell proliferation. Dev Cell 10:397-405.

2.   Giraud S, Claire B, Eugene M, Debre P, Richard F, and Barrou B. (2007) A new preservation solution increases islet yield and reduces graft immunogenicity in pancreatic islet transplantation. Transplantation. 83:1397-400 .

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 46:1120-3.

4.   Olack B.J., Swanson C.J., Howard T.K., and Mohanakumar T. (1999) Improved method for the isolation and purification of human islets of langerhans using Liberase enzyme blend. Hum Immunol 60:1303-9.

5.   Gotoh M, Maki T, Satomi S, Porter J, Bonner-Weir S, O’Hara CJ, and Monaco AP (1987) Reproducible high yield of rat islets by stationary in vitro digestion following pancreatic ductal or portal venous collagenase injection. Transplantation. 43:725-30.

6    Carter JD, Dula SB, Corbin KL, Wu R, and Nunemaker CS (2009) A practical guide to rodent islet isolation and assessment. Biological Procedures Online. Proc. Online 11:3-31.

7    Li D-S, Yuan Y-H, Tu H-J, Liang Q-L and Dai L-J (2009) A protocol for islet isolation from mouse pancreas. (2009) Nature Protocols 4:1649-52.