Bob McCarthy
For those who attended VitaCyte’s last webinar, “Is there an optimal enzyme dose for human islet isolation” on June 18, 2026, thank you. All but two attendees listened to the entire 60-minute webinar, and the majority stayed for the 30-minute Q&A session. For those who missed the webinar, a recording is posted here.
I found Doug O’Gorman’s presentation analyzing the results of 1094 human islet isolations at the University of Alberta in Edmonton very interesting, as it provided additional insights into the effects of enzymes on islet yield and function. Below, I summarize four notable observations he made during his presentation.
First, Doug states on slide 41 “The use of Wünsch assay as the primary dosing metric is fundamentally flawed.” The dose of Liberase™ HI Purified Enzyme Blend recommended for human islet isolation was based on Wünsch activity, since it was the most reliable collagenase activity measurement. Improved knowledge of Clostridium histolyticum structure-function shows three major limitations when using Wüncsh activity to determine collagenase dose.
- It only measures class II collagenase activity; the specific activity of class I collagenase measured by Wünsch units per mg protein (WU/mg) is > 50 fold lower than class II
- The Wünsch (or FALGPA) activity measures cleavage of a collagen-specific peptide; the only requirement for activity is a functional collagenase catalytic domain
- Wünsch activity does not correlate with collagen-degradation activity, which is the true measure of collagenase function; an earlier study showed no correlation of Wünsch activity to islet yield.
Second, Doug’s analysis on slide 27 showed “there was no significant effect of enzyme type on yield per gram after controlling for organ size.” The enzyme referred to is collagenase. His statement is consistent with the proposed mechanism of action for collagenase-protease-mediated tissue dissociation as discussed in an earlier blog post and shown in the medical animation video here. The model proposes that, as long as collagenase’s collagen-degrading activity (CDA) is in excess, collagenase thins out the collagen fibril jungle within the extracellular matrix (ECM) and loosens the ECM structure, leading to exposure of the protease-sensitive sites on ECM cell anchoring proteins cut by neutral proteases.
Third, Doug’s analysis on slides 31 & 32 shows that donors under 45 have nearly 2x as many embedded islets as donors over 45. Increasing neutral protease activity does not increase islet yields.
The collagenase-protease mixtures used in these studies contained a single neutral protease. Doug noted that more work is needed on this issue, as the dose was uncontrolled in younger donor organs. Balamurugan Appakalai, in an earlier VitaCyte webinar, updated his results on isolating islets from young human donors, showing that the islet isolation process needed to be modified when using a higher neutral protease dose to improve islet yields from these organs.
I also believe that using multiple proteases will improve islet yields. At the beginning of the webinar, I showed a table from the initial report describing the use of Liberase HI for human islet isolation. Here, version 1 of Liberase contained 12,000 BAEE U of clostripain in addition to purified thermolysin and collagenases. These results, summarized in an earlier blog post, showed that when tissue digestions were performed on 9 organs from donors < 25 and 17 > 25, the total islet equivalent counts (IEQ) from older donors were nearly twice those from younger donors. By contrast, when results were expressed as IEQ/g tissue, there was no difference in islet recovery.
The last and most interesting observation follows up on an earlier report from the University of Alberta group that showed that lowering the dose of purified natural or recombinant collagenase (16 WU/g tissue) did not affect islet yields compared with a standard dose of natural purified collagenase (20 WU/g tissue). Slide 39 shows that islets isolated with the lower dose of either collagenase gave higher in vitro glucose-stimulated insulin responses (GSIR) as measured by peak insulin, insulin area under the curve, stimulated metabolic rate, and stimulated metabolic output. Islets isolated with recombinant collagenase were the only ones that showed a significant difference compared to those isolated with the standard dose of collagenase.
I have no explanation as to why a lower dose of collagenase improved in vitro islet function. These differences between the low-dose purified natural and recombinant collagenase could be due to an unidentified contaminant in the natural collagenase or to the greater effectiveness of recombinant collagenase in recovering functional human islets.
The information shared at this webinar marks the first time there has been a focused discussion on enzyme dose and human islet yield. It explains why nearly all attendees listened to the entire webinar.
Balamurugan Appakalai (Bala) from the Wendy Novak Diabetes Institute, a part of Norton Healthcare in Louisville, KY, will give a webinar on his research determining the effect of enzyme dose on islet yield and function on September 17, 2026. Bala oversaw the design of experiment study performed at the University of Minnesota, which was summarized in my last blog post and by Mike Green in the June 18th webinar. Bala extended these results by showing that a low dose of recombinant collagenase (12 WU/g tissue) was as effective as the standard dose of natural purified collagenase (20 WU/g) for human islet isolation. Moreover, reducing the dose of natural purified collagenase to 12 WU/g resulted in 50% recovery of islets compared with isolations using the same dose of recombinant collagenase.