New insights for using purified enzymes in human islet isolation

The introduction of Liberase HI Purified Enzyme Blend in 1994 to improve the consistency of enzymes used to recover human pancreatic islets for subsequent transplantation contributed to the growth of clinical islet transplantation.(1)  Liberase HI set a benchmark for comparing the performance of all subsequent products used for this application.(2, 3) A series of reports by Balamurugan et al., provided deeper insights into the enzyme compositions required to increase human islet yields.(3-6) The first two reports showed the importance of intact class I (C1) collagenase and the appropriate neutral protease enzyme for isolating human islets.(3, 4)  In 2016, they reported that a wide range of purified, recombinant C1 to class II (C2) collagenase mixed with a fixed dose of BP Protease, gave high islet yields (≈ 5000 IEQ/g pancreas).(5) Their most recent publication in American Journal of Transplantation(6) presents data that supports and extends these earlier observations by showing:

  • purified recombinant collagenase is more efficient for recovery of human islets than purified natural collagenase;
  • BP Protease (a Dispaseä equivalent enzyme) is as effective as Clostridium histolyticum neutral protease to recover human islets;
  • decreasing the amount of exogenous enzyme used in the islet isolation procedure appears to improve islet function.

This blog post reviews the key findings above and comments on their implications to improve the yield of functional human islets used for subsequent transplantation.

Recombinant collagenase is a superior enzyme for human islet isolation

All isolations in this report(6) used a fixed amount of BP Protease activity per g trimmed pancreas (23,400 neutral protease units) that enabled direct comparison of the effectiveness of different doses of purified, natural or recombinant Clostridium histolyticum collagenase to isolate human islets. The results from these isolations compared islet yields isolated with a low (12 Wunsch units – WU) or high (20 WU) dose of collagenase per g pancreas. These data are summarized below:

Enzyme C1:C2 ratio WU/g pancreas n IEQ/g pancreas

± one std dev

% undigested pancreas

± one std dev

Natural collagenase 60:40 20 8 4946 ± 974 20.1 ± 9
12 5 2582 ± 925* 37.8 ± 3*
Recombinant collagenase 30:70 20 5 5535 ± 830 15.7 ± 1
12 7 5455 ± 918* 16.7 ± 5*
WU = Wunsch U,   n = number of isolations,  *P < 0.05

 

The enzyme mixtures used for these isolations had different C1:C2 ratios. The 60:40 C1:C2 ratio used for purified, natural collagenase enzyme product (VitaCyte’s CIzyme Collagenase HA) was comparable to those used in Liberase HI. In contrast, recombinant collagenase contained a 30:70 C1:C2 ratio. This difference was unlikely to impact islet yields for two reasons. Similar human islet yields (≈ 5000 IEQ/g pancreas) were obtained when recombinant C1:C2 at ratios of 55:45, 43:57, 38:62, and 27:73 mixed with a fixed dose of BP Protease activity (23,400 NP U/g pancreas) were used to isolate islets from the body-tail of a human pancreas.(5) The 30:70 C1:C2 mixture used for islet isolation contained about 40% of the amount typically used for natural collagenase isolation.

Comparison of the results from isolations using low dose, recombinant collagenase to those using the same dose of natural, purified collagenase showed significant differences in islet yield. Low dose purified, natural collagenase gave ≈ 50% reduction in islet yield and ≈ 1.9 fold increase in undigested tissue when compared to those isolations that used a high dose of purified natural or low or high dose of recombinant collagenase.(6) This observation is the first report directly showing the effect of suboptimal dose of purified natural collagenase on human islet yield.

The reasons for the difference in the effectiveness are unknown since there are only minor changes in the amino acid sequence between these two enzyme preparations:  4 and 13 amino acid differences for C1 and C2, respectively.(5) One conclusion from these results is not all sources of collagenase are equivalent. These results are in contrast to those in an earlier report evaluating the effectiveness of recombinant collagenase. Here, the effectiveness of purified recombinant or natural collagenase 60:40 C1:C2 mixtures combined with the same dose of thermolysin used in Liberase HI were compared for their efficiency to isolate human islets. The only significant difference between the two groups was the higher islet index (i.e., IEQ/islet number) obtained when recombinant collagenase-thermolysin was used to isolate islets.(7)

BP Protease is as effective as NB Protease in human islet isolation

Earlier reports indicated the superiority of using a “milder” neutral protease (C. histolyticum neutral protease, Nordmark-Serva NB Protease) in place of the stronger thermolysin enzyme used in the Liberase HI formulation.(4)  Balamurugan, et al., directly compared human islets isolated from a split pancreas using a fixed collagenase dose (i.e., Collagenase HA) and optimal neutral protease activities. The only difference in these mixtures was the use of thermolysin or NB Protease. He termed the Collagenase HA-NB Protease mixture as the new enzyme mixture (NEM). The NEM gave significantly higher post purification islet yields and an increased percentage of islets with diameters > 200 μm than those islets isolated from the collagenase-thermolysin mixture.

Purified BP Protease (a Dispaseä equivalent enzyme) is purified from Paenibacillus polymyxa culture supernatants that do not contain any animal proteins. This protein belongs to the M4 protease family whose members include thermolysin and C. histolyticum neutral protease.(8) Although all members of this family have the same specificity for cleavage at the amino-terminal side of hydrophobic amino acids, they have different selectivity for different amino acids. For example, a study comparing the effectiveness of thermolysin or Dispaseä to remove epithelial cells from cryopreserved human amniotic membranes showed that each enzyme gave a different degradation pattern of the extracellular matrix proteins associated with this tissue as detected by immunohistochemical methods.(9) Treatment with Dispase eliminated staining of type VII collagen, laminin 5, and integrins a6 and b4 but retained staining of collagen type IV.  By contrast, there was no loss of staining of these proteins after treatment with thermolysin. The importance of citing this study is to show differences in the ability of each protease to digest extracellular matrix proteins for this specific biological material. This observation may or may not apply to similar studies since thermolysin and Dispase are routinely used to recover hepatocytes or islets from rodent or human tissue.

Comparison of the effectiveness of purified recombinant collagenase-BP protease enzyme mixtures or NEM to isolate islets from the body-tail of human pancreas are summarized below.(5) Both groups used equivalent amounts of neutral protease activity per g pancreas in the islet isolation process. The results from these isolations are summarized below.

Enzyme mixture n IEQ per g pancreas ± 1 std dev
rC1/rC2 + BP Protease 12 5209 ± 1809
Collagenase HA + NB Protease 6 3615 ± 1809
IEQ = Islet equivalent number

 

 

 

 

The results from isolations using different amounts of recombinant C1 and C2 collagenase were aggregated together for two reasons. There was no difference in the performance of the four enzyme mixtures noted above to recover human islets. Moreover, the collagenase concentrations were below those used for islet isolation using natural, purified collagenase. The range in the mass of recombinant collagenase used in these isolations ranged from 2.09 to 3.75 mg/g tissue. In contrast, the Collagenase HA in the NEM contained about 4.5 mg/g pancreas.(5)

The results above show that the recombinant collagenase-BP Protease mixture gave a higher mean islet yield, but there was no statistically significant difference between these two groups.

The table below summarizes all the human islet isolations performed in the last two reports.(5, 6) All isolations used excess amounts of purified natural or recombinant collagenase combined with a fixed dose of BP Protease activity per g pancreas (23,400 NP U).

Enzyme mixture Reference WU/g pancreas mg collagenase/g pancreas n Islet yield/g pancreas

± one std dev

rC1 + rC2 (5) 12 or 20 2.09-3.75 12 5209 ± 1809
rC1 + rC2 (6) 12 2.09 7 5455 ± 918
rC1 + rC2 (6) 20 3.48 5 5535 ± 830
Collagenase HA (6) 20 4.41 8 4946 ± 974
rC1 or rC2 = recombinant collagenase   Collagenase HA = purified natural collagenase with 60:40 C1:C2 ratio

 

 

The average islet yield was approximately 5,000 IEQ/g pancreas after digest of 32 human research pancreata. There were no failed isolations.

Decreasing amounts of exogenous enzyme in islet isolation appears to improve islet function

An unexpected benefit of using lower doses of collagenase for human islet isolation may be improved functional activity. Comparison of glucose-stimulated insulin release from islets isolated with purified, low dose, recombinant collagenase gave significantly higher basal insulin secretion after 48 hours of culture than those islets isolated with purified, high dose, natural collagenase. There was no significant difference in basal insulin secretion when islets isolated with low or high dose recombinant collagenase or with low dose natural collagenase.(6)

Basal insulin secretion after 48 hours of culture per 50 islets (μU/mL)

Collagenase Enzyme Mixture Low dose 12 WU/g pancreas High dose 20 WU/g pancreas
Recombinant 30:70 C1:C2 2391 ± 1342* 2097 ± 976
Natural 60:40 C1:C2 2007 ± 1012 1778 ± 978*
*Significant difference P < 0.05

 

Additional experiments determined the effect of pre-treating purified human islets with either low or high dose purified, recombinant collagenase or purified, natural collagenase for 30 min, to control blood glucose in nude mice. Here, pre-treated islets were implanted under the kidney capsule and challenged with high glucose and an intraperitoneal glucose tolerance test (IPGTT) performed by collecting blood over a 90 min period, followed by analysis of blood for glucose and insulin. These results showed that the sham-treated control gave the best glycemic control. However, comparison of IPGTT results from diabetic mice that received islets pretreated with low dose recombinant collagenase showed significantly better glycemic control and secreted a greater amount of insulin during the analysis period than other pretreated islet preparations (i.e., high dose recombinant, or low or high dose natural collagenase). (6)

Implications

The results from the two recent reports from Balamurugan and colleagues(5, 6) reinforces the general belief that enzyme composition and dose are critical process parameters for manufacturing human islets for clinical transplantation. These reports concluded that

  • not all collagenase preparation are equivalent;
  • BP Protease when used with an optimal dose of natural or recombinant collagenase, consistently gave high islet yields (average 5000 IEQ/g pancreas) from human research pancreata;
  • reducing the dose of collagenase may increase islet function, potentially impacting the functional performance of the engrafted islets.

The implications of these conclusions indicate that dose-response curves should be performed to validate the purified collagenase and protease enzymes used in the islet isolation procedure.  Several independent lots of product should be used for validation to ensure the consistency of the results.

References

  1. Second Annual Report Bethesda, MD: Collaborative Islet Transplant Registry 2005. www.citregistry.org (accessed 3 April 2018).
  2. Bucher P, Mathe Z, Morel P, Bosco D, Andres A, Kurfuest M, et al. Assessment of a novel two-component enzyme preparation for human islet isolation and transplantation. Transplantation. 2005;79(1):91-7.
  3. Balamurugan AN, Breite AG, Anazawa T, Loganathan G, Wilhelm JJ, Papas KK, et al. Successful human islet isolation and transplantation indicating the importance of class 1 collagenase and collagen degradation activity assay. Transplantation. 2010;89(8):954-61.
  4. Balamurugan AN, Loganathan G, Bellin MD, Wilhelm JJ, Harmon J, Anazawa T, et al. A new enzyme mixture to increase the yield and transplant rate of autologous and allogeneic human islet products. Transplantation. 2012;93(7):693-702.
  5. Balamurugan AN, Green ML, Breite AG, Loganathan G, Wilhelm JJ, Tweed B, et al. Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation. Transplantation Direct. 2016;2(1):e54.
  6. Loganathan G, Subhashree V, Breite AG, Tucker WW, Narayanan S, Dhanasekaran M, et al. Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield. American Journal of Transplantation. 2018;18(2):478-85.
  7. Brandhorst H, Brandhorst D, Hesse F, Ambrosius D, Brendel M, Kawakami Y, et al. Successful human islet isolation utilizing recombinant collagenase. Diabetes. 2003;52(5):1143-6.
  8. van den Burg B, Eijsink V. Thermolysin and related Bacillus metalloproteases. In: Barrett AJ, Rawlings ND, J.F. W, editors. Handbook of Proteolytic Enzymes. 1. Second ed. Boston: Elsevier Academic Press; 2004. p. 374-87.
  9. Hopkinson A, Shanmuganathan VA, Gray T, Yeung AM, Lowe J, James DK, et al. Optimization of amniotic membrane (AM) denuding for tissue engineering. Tissue Engineering Part C, Methods. 2008;14(4):371-81.

 

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