Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

Balamurugan, Appakalai N.; Green, Michael L.; Breite, Andrew G.; Loganathan, Gopalakrishnan; Wilhelm, Joshua J.; Tweed, Benjamin; Vargova, L.; Lockridge, Amber; Kuriti, Manikya; Hughes, Michael G.; Williams, Stuart K.; Hering, Bernhard J.; Dwulet, Francis E.; McCarthy, Robert C.

doi:10.1097/txd.0000000000000563

Cited by 23 publications

(34 citation statements)

References 26 publications

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“…36 Our result showed that the BM proteins were high in YDP when compared to ODP. In a subsequent study, 18 we reported the effectiveness recombinant rC1/rC2 on human islet isolation. In one of our previous study, 17 we identified an important role of C1/C2 collagenases in effective digestion of collagen proteins in that, intact C1 collagenase is critical for obtaining a higher islet yield.…”

Section: Discussionmentioning

confidence: 99%

“…23 In subsequent studies, islet isolations performed using whole pancreas from YDP used one of three collagenase-protease enzyme mixtures from VitaCyte, Roche, or SERVA (Table 1). 23 In subsequent studies, islet isolations performed using whole pancreas from YDP used one of three collagenase-protease enzyme mixtures from VitaCyte, Roche, or SERVA (Table 1).…”

Section: Enzyme Combinations Used For Human Islet Isolationsmentioning

confidence: 99%

“…The transplanted graft success rate in nude mice was 88% for VitaCyte (7 of 8), 75% for Roche (6 of 8), and 83% for SERVA (5 of 6) ( Figure 3C). SEC, standard enzyme combination (1,5,9,10,14,18); ICC, increased collagenase combination (2,6,7,11,15,16); IPC, increased protease combination (3,4,8,12,13,17). 29 Histopathological examination of a successful human islet graft retrieved after 30 days showed insulin staining (brown) indicating viable islets ( Figure 3D).…”

Section: Whole Pancreas Islet Isolation Outcomementioning

confidence: 99%

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Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Loganathan

Venugopal

Narayanan

et al. 2019

American Journal of Transplantation

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Human islet isolation from young donor pancreases (YDP) utilizing the current purified standard dose of collagenase-protease enzyme mixtures often results in the release of a high percentage of mantled islets. Mantled islets are those surrounded by exocrine tissue and are difficult to purify by density gradient centrifugation, leading to poor islet recovery. Based on difference in extracellular matrix, and total collagen content between YDP and old donor pancreas (ODP, > 35 Y) led us to compare results from islet isolation using increased collagenase combination (ICC) or increased protease combination (IPC), to the standard enzyme combination (SEC) in a "trisected" pancreas model to overcome the donor-to-donor variability. These results showed a reduced percentage of mantled islets (17% ± 7.5%) and higher postpurification islet recovery (83.8% ± 5.6%) with IPC. Furthermore, these results were confirmed in 13 consecutive whole pancreas islet isolations utilizing IPC from VitaCyte, Roche, or SERVA collagenase-protease enzyme mixtures. Results obtained from in vitro and in vivo islet functional assessment indicated that islets isolated using IPC retained normal islet morphology, insulin secretion, and the ability to reverse diabetes after transplantation in diabetic nude mice. This is the first report utilizing trisected pancreas to assess the effectiveness of different enzyme combinations to improve islet recovery from young donor pancreases.

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Section: Discussionmentioning

confidence: 99%

Section: Enzyme Combinations Used For Human Islet Isolationsmentioning

confidence: 99%

Section: Whole Pancreas Islet Isolation Outcomementioning

confidence: 99%

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Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Loganathan

Venugopal

Narayanan

et al. 2019

American Journal of Transplantation

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“…In addition, a recent report by Balamurugan et al 18 has demonstrated that enzyme activity levels can be reduced by 50% to 60% using standard human islet techniques with “no significant main effect of Wunsch activity or CDA for any parameter measured.” Although significant differences exist between our static digestion methodology and the Ricordi method that is standard practice in human islet isolation laboratories, these findings along with the data presented here suggest that the static digestion method and/or reduced enzyme activity levels merit further evaluation for use with human pancreas.…”

Section: Discussionmentioning

confidence: 99%

Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function

Holdcraft

Green

Breite

et al. 2016

Transplantation Direct

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BackgroundHuman allogeneic islet transplantation for treatment of type 1 diabetes provides numerous clinical benefits, such as fewer episodes of hypoglycemic unawareness and tighter control of blood glucose levels. Availability of human pancreas for clinical and research use, however, is severely limited. Porcine pancreas offers an abundant source of tissue for optimization of islet isolation methodology and future clinical transplantation, thereby increasing patient access to this potentially lifesaving procedure.MethodsPorcine islet isolations were performed using varying amounts of collagenase (7.5, 3.75, or 2.5 Wunsch units per gram tissue) and neutral protease activity (12 000, 6000, or 4000 neutral protease units per gram tissue) and perfusion volumes (1.7 or 0.85 mL/g tissue) to assess their effects on isolation outcomes. Retention of dissociative enzymes within the pancreas during perfusion and digestion was evaluated, along with distribution of the perfusion solution within the tissue.ResultsReducing enzyme usage by as much as 67% and perfusion volume by 50% led to equally successful islet isolation outcomes when compared with the control group (48 ± 7% of tissue digested and 1088 ± 299 islet equivalents per gram of pancreas vs 47 ± 11% and 1080 ± 512, respectively). Using margin-marking dye in the perfusion solution to visualize enzyme distribution demonstrated that increasing perfusion volume did not improve tissue infiltration.ConclusionsCurrent protocols for porcine islet isolation consume excessive amounts of dissociative enzymes, elevating cost and limiting research and development. These data demonstrate that islet isolation protocols can be optimized to significantly reduce enzyme usage while maintaining yield and function and thus accelerating progress toward clinical application.

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“…In our recent study, 19 we successfully manufactured purified rC1 and rC2 and assessed 4 different collagenase formulations to recover islets from human pancreases (n = 12, 3 per group). Varying amounts of rC1 and rC2 collagenase activity, supplemented with a fixed amount of P. polymyxa NP activity (BP-Protease [VitaCyte LLC, Indianapolis, IN], a dispase-equivalent NP), were tested using a statistically designed experiment in a split-pancreas model.…”

mentioning

confidence: 99%

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield

Loganathan

Venugopal

Breite

et al. 2018

American Journal of Transplantation

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A high number of human islets can be isolated by using modern purified tissue dissociation enzymes; however, this requires the use of >20 Wunsch units (WU)/g of pancreas for digestion. Attempts to reduce this dose have resulted in pancreas underdigestion and poor islet recovery but improved islet function. In this study, we achieved a high number of functional islets using a low dose of recombinant collagenase enzyme mixture (RCEM-1200 WU rC2 and 10 million collagen-degrading activity [CDA] U of rC1 containing about 209 mg of collagenase to digest a 100-g pancreas). The collagenase dose used in these isolations is about 42% of the natural collagenase enzyme mixture (NCEM) dose commonly used to digest a 100-g pancreas. Low-dose RCEM was efficient in digesting entire pancreases to obtain higher yield (5535 ± 830 and 2582 ± 925 islet equivalent/g, P < .05) and less undigested tissue (16.7 ± 5% and 37.8 ± 3%, P < .05) compared with low-dose NCEM (12WU/g). Additionally, low-dose RCEM islets retained better morphology (confirmed with scanning electron microscopy) and higher in vitro basal insulin release (2391 ± 1342 and 1778 ± 978 μU/mL; P < .05) compared with standard-dose NCEM. Nude mouse bioassay demonstrated better islet function for low-dose RCEM (area under the curve [AUC] 24 968) compared with low-dose (AUC-38 225) or standard-dose NCEM (AUC-38 685), P < .05. This is the first report indicating that islet function can be improved by using low-dose rC1rC2 (RCEM).

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Identifying Effective Enzyme Activity Targets for Recombinant Class I and Class II Collagenase for Successful Human Islet Isolation

Cited by 23 publications

References 26 publications

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Improved recovery of human islets from young donor pancreases utilizing increased protease dose to collagenase for digesting peri-islet extracellular matrix

Optimizing Porcine Islet Isolation to Markedly Reduce Enzyme Consumption Without Sacrificing Islet Yield or Function

Beneficial effect of recombinant rC1rC2 collagenases on human islet function: Efficacy of low-dose enzymes on pancreas digestion and yield

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