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Presenter: J., Abouaish, Minneapolis, USA
Authors: J. Abouaish, M. Graham, P. Pakala, G. Loganathan, M. Tiwari, T. Yuasa, K.K. Papas, D.E.R. Sutherland, R.C. McCarthy, B.J. Hering, A.N. Balamurugan
A new purified enzyme blend for non-human primate islet isolation
J. Abouaish1, M. Graham1, P. Pakala1, G. Loganathan1, M. Tiwari1, T. Yuasa1, K.K. Papas1, D.E.R. Sutherland1, R.C. McCarthy2, B.J. Hering1, A.N. Balamurugan1
1 Schulze Diabetes Institute, Dept. of Surgery, University of Minnesota, Minneapolis, USA; 2 VitaCyte, Indianapolis, USA
Background: Nonhuman primates (NHPs) are essential for pre-clinical safety and efficacy studies in islet allotransplantion. In view of ethical considerations and the limited availability of NHPs, it is critical that a sufficient number of high quality islets are obtained from every processed donor pancreas. After Liberase-HI (LHI) enzyme was discontinued, finding another source of enzymes capable of producing consistently high islet yields in NHPs was difficult. We optimized the ideal dose and ratio for a purified enzyme blend (PEB) composed of VitaCyte’s CIzyme™ Collagenase-MA and CIzyme™ Thermolysin to maximize islet yield.
Methods: Islets were isolated from male rhesus macaques (Macaca mulatta) (n=20) using a modified Ricordi method. The pancreas was infused with 750 Wunsch units (6.15 million CDA units) of collagenase-MA and 6mg (1.69 million thermolysin activity units) of Thermolysin. Islets were purified using a COBE 2991 cell processor and continuous iodoxonal gradients, and cultured (91±1hrs) before transplantation. The results from these isolations were compared to historical LHI isolations.
Results: Donor and isolation results are described in the table. The donors had an average body weight of 12.3±2.8kg and an average pancreas weight of 27.0±10.2g. The average islet yields obtained with the PEB were 77,225±31,195 after digestion, 62,079±22,667 post-purification, and 69,373±24,858 IEQ post culture. The percent recovery after COBE purification was 84.2±33.9%. The average transplanted IEQ/kg was 8,080±2,892 IEQ/kg. Significant differences were noted when PEB data was compared with historical LHI data: the digestion time was significantly shorter in the VCT isolations (11.3±1.8, 18.0±5.3min, p<0.01) and the post digest quality score was higher for PEB isolated islets (8.5±0.6, 7.8±1.0, p=0.01).
Conclusions: In the absence of LHI, the optimal dose of the PEB was identified. The PEB consistently produced a high islet yield with results comparable to LHI, and is an effective enzyme blend for NHP islet isolation.
Disclosure: Author R. C. McCarthy is a co-founder and shareholder of VitaCyte LLC
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