2017 - IPITA


Islet Isolation, Culture & Preconditioning 1

14.11 - Exogenous Clostripain Improves Human Pancreas Islet Digestion Without Impacting Islet Function

Presenter: Gregory, Szot, San Francisco, United States
Authors: Gregory L Szot, Kate E Smith, Nicolas D Price, Craig S Webber, Vi Dang, Andrew M Posselt, Klearchos K Papas


Exogenous Clostripain Improves Human Pancreas Islet Digestion Without Impacting Islet Function

G. Szot1, K. Smith2, N. Price2, C. Webber3, V. Dang1, A. Posselt1, K. Papas2.

1Division of Transplant, University of California, San Francisco, USA, ; 2Department of Surgery, University of Arizona, Tucson, USA, ; 3Department of Physiology, University of Arizona, Tucson, USA,

Introduction: Digestion of a human pancreas for the isolation of islets requires Collagenase and Neutral Protease. It has been shown that increased amounts of Neutral Protease (NP) or tryptic-like-protease activity (TLA) can significantly improve islet yields. Can the addition of Clostripain, a trypsin-like-protease and neutral protease, function similarly? Here we evaluate the effects of exogenous Serva Clostripain AF, in combination with TLA-Free Serva AF-1 Collagenase and Neutral Protease on islet processing.

Methods: Standard islet isolations from deceased donor pancreata were performed at the University of California San Francisco (UCSF) using Serva NB1 Collagenase and Neutral Protease (n=4) or at University of Arizona (UA) using Serva Premium Collagenase and Neutral Protease (NB1/P) (n=2); and Serva AF-1 Collagenase and Neutral Protease (AF1) with the addition of 100 U (n=1); or with the addition 400 U (n=2) Clostripain (CP) at UCSF. Islet characterization viability measurements were performed by Oxygen Consumption Rate normalized to DNA (OCR/DNA) assay, higher OCR/DNA values indicates higher viability. Functional measurements were conducted using a dynamic perfusion system. 100 IEQs (x3) were exposed to basal glucose (2.8mM) flow for 10 minutes, and then stimulated for 40 minutes with 16.7 mM glucose. Samples were analyzed for secreted hormones [Insulin, C-Peptide, Pancreatic Polypeptide, and Glucagon] by Multiplex Analysis at the University of Arizona.

Results: Digestion/recirculation time was reduced from 14.7 ±2.2 minutes in the NB1/P group to 11.5 ±0.71 minutes in the AF1+ 400U CP; the single isolation of AF1+ 100U CP reduced time to 14 minutes. Pre-purification islet counts were: 490,146 IEQ ±152,191(NB1/P); 653,475 IEQ ±71,735 (AF1+ 400U CP), an increase of 25%; and 475,200 IEQ (AF1+ 100U CP). Post-purification islet counts were 381,103 IEQ ±73,649 (NB1/P), 510,150 IEQ ±78,842 (AF1+ 400U CP) and 630,700 IEQ (AF1+ 100U CP). An improvement in islet yield compared to NB1 alone of 25% and 39% respectively. Clostripain also seemed to reduce the number of embedded/mantled islets, even in young pancreata.

Each islet group had a higher OCR/DNA value, Table 1, than historical clinical islet average of 125 ±28 nmol O2/min*mg DNA. Average hormone secretion indices (ratio of High/Low glucose stimulation) measured for each group and hormone are reflected in the Table 1.

Table 1

 

OCR/DNA

 

Insulin

 

C-Peptide

Pancreas

Polypeptide

 

Glucagon

 

(nmol O2/min*mg DNA)

(pg/ng DNA)

Collagenase NB1/P (n=6 OCR)(n=4 hormone)

149.06  +27.15

4.77 +4.42

4.27 +4.20

1.33 +0.390

0.697 +0.103

Collagenase AF1+ 400U CP (n=2)

161.25 +15.77

3.22 +0.10

3.20 +0.00

1.35 +0.494

0.825 +0.035

Collagenase AF1+ 100U CP (n=1)

134.7

2.2

1.83

0.95

0.57

Conclusions: Addition of Clostripain reduced total digestion/recirculation times, increased islet yields, and reduced contaminating acinar tissue. Moreover, Clostripain did not compromise Islet function or their ability to consume oxygen or to secrete glucose stimulated insulin, C-peptide and pancreas polypeptide. The Collagenase NB1/P group showed a slightly greater insulin and C-peptide secretion; however the sample size is small in this study and requires additional evaluation. We conclude that the controlled addition of Clostripain to pancreas digestion may reduce the amount of potentially toxic/dying acinar, improve islet yields, while preserving islet function and viability.


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