Bob McCarthy
Balamurgan Appakalai (Bala) from the Wendy Novak Diabetes Institute in Louisville, KY, will present a webinar on “Low-dose collagenase for human islet isolation: Recombinant vs natural collagenase” on September 17, 2026, at 11 AM EDT. The webinar will review his research evaluating the effectiveness of a low dose of recombinant collagenase in recovering human islets. This dose was the lowest amount of collagenase used in an earlier enzyme dose study published in Transplantation Direct. Mike Green reviewed additional details of the experimental design and results from this report in the last VitaCyte webinar.
Loganathan et al. compared the effectiveness of 30:70 recombinant class I:class II (rC1:rC2) enzyme mixture supplemented with 23,400 neutral protease U (NP U) of BP Protease (#003-1000) against VitaCyte’s natural purified collagenase (Collagenase HA, #001-1000) containing a 60:40 C1:C2 mixture at 12 and 20 Wunsch U per g tissue. These results are summarized in the table below
Summary of Results from Loganathan et al AJT 18:478-485, 2018
WU/g = Wunsch U per g of trimmed pancreatic tissue
*All enzyme mixtures supplemented with BP Protease @ 23,400 neutral protease U/ g tissue
These results confirmed the earlier observation that reducing the collagenase dose by 40% did not affect human islet yields. Reducing the dose of purified natural collagenase to 12 WU/g tissue resulted in a 50% loss of islet yield. Moreover, basal insulin release from islets isolated with low-dose recombinant collagenase was significantly better than from islets isolated with a 20 WU dose, commonly used for human islet isolations today.
The results from the DOE dose study led VitaCyte to sell rCollagenase HI (001-4010) at a dose that was the midpoint of the 4 different doses used in the earlier study: 1600 WU of rC2 and 15 million of rC1 per bottle, an amount sufficient to digest 100 g pancreas using a 50:50 rC1:rC2 mixture at a dose of 16 WU of rC2 and 150,000 CDA U of rC1 per g tissue.
The University of Alberta in Edmonton used the rCollagenase HI product for human islet isolations for clinical islet transplantation. In the last webinar, Doug O’Gorman analyzed results from 1094 human islet isolations to answer the question: Is there an optimal enzyme dose?
Included in this summary were results from two abstracts presented at the 2021 virtual IPITA meeting and at the IPITA-IXA-CTRMS Joint Congress meeting in San Diego in October 2023. The first abstract showed that the rCollagenase HI-BP Protease mixture used at a dose of 16.8 WU and 158,000 CDA U per g tissue (n= 49) was as effective in recovering islets as collagenase-thermolysin mixtures from Roche’s MTF kit (n= 229) or VitaCyte’s Collagenase HA-Thermolysin mixtures (n= 109). Here, all three enzyme mixtures gave equivalent post-purification total islet yields and percent viability. The enzyme mixtures from the three products used in the study had comparable neutral protease activity when Roche’s Thermolysin activity units were converted to VitaCyte’s neutral protease units. The main difference was the collagenase dose, as determined by Wunsch activity. Here, the natural purified collagenase was used at a dose between 28-32 WU/g tissue. By contrast, the recombinant collagenase was used at a dose of 16-18 WU/g tissue.
Notably, human islets isolated with the rCollagenase HI-BP Protease mixture yielded significantly higher recoveries of purified islets after short-term culture than islets isolated with natural purified collagenase and BP Protease. Consequently, a significantly higher number of islets were transplanted per kg body weight when the same comparison was made with islets isolated with natural purified collagenase.
Another benefit of using human islets isolated with a lower dose of recombinant collagenase was a significantly higher insulin response in a glucose-stimulated insulin release (GSIR) assay using a perifusion device.
Comparison of the Beta 2 Score or percent insulin reduction in patients receiving islets from these three groups showed no significant difference in these clinical measurements.
In the second abstract, O’Gorman et al. showed that similar human islet yields were obtained when comparing results from islets isolated using a high-dose Roche MTF kit (HD-MTF: 31.5-32.5 WU/g tissue), a low-dose MTF (LD-MTF: 17-17.5 WU/g), or rCollagenase HI (LD rHI: 16.5 WU/g). As noted above, comparable neutral protease activities of Thermolysin (Roche) or BP Protease (VitaCyte) were used for these isolations.
Several key findings are summarized in the table below.
Several notable findings from this report are:
- Longer digestion times with the LD-MTF and LD-rHI mixtures when compared to the HD-MTF mixture
- In the GSIR assay, islets isolated using the LD-MTF and LD-rHI had higher stimulated metabolic rates and metabolic output when compared to islets isolated with HD-MTF, with significant differences detected when the results from the LD-rHI and HD-MTF were compared
- No significant difference in recovery of islets after culture, but a significantly higher percentage of the islet isolations using LD-rHI were transplanted when compared to LD-MTF islet preparations
The results summarized above from Bala’s lab and O’Gorman et al. clearly indicate that decreasing the collagenase dose used for human islet isolation improves in vitro islet function, as measured by GSIR assays. O’Gorman et al.’s results show that when 17 WU/g of rCollagenase HI is used for human islet isolation, a higher percentage of these preparations can be used for transplantation.
Several open questions for discussion at Bala’s webinar on September 17 are:
- What is the lowest dose of collagenase that can be used for clinical islet isolations?
- Do you see any noticeable differences between islets isolated with recombinant or natural collagenase?
- Is there an explanation for the superior performance of the recombinant collagenase versus the natural purified collagenase, especially in the higher percentage of islets used for transplant?
- What is the predictive value of in vitro GSIR assays since O’Gorman et al. results indicate there is no difference in clinical outcomes when islets were isolated with either HD natural purified collagenase vs. low-dose recombinant collagenase?