P-202

Biological characterization of isolated juvenile porcine islets as a potential source of tissue for xenotransplantation.

B.P. Weegman¹, M.J. Taylor², S.C. Baicu², R. Pongratz³, K.R. Mueller¹, T.D. O'Brien⁴, G. Cline³, J. Wilson⁵, K.K Papas^1
1 University of Minnesota, Department of Surgery, Minneapolis, MN, USA; ² Cell and Tissue Systems Inc., N. Charleston, SC, USA; ³ Yale University, New Haven, CT, USA; ⁴ University of Minnesota, Department of Veterinary Population Medicine, Saint Paul, MN, USA; ⁵ Wilson Wolf Manufacturing, New Brighton, MN, USA

Objective: Limited availability of donor human pancreata means that clinical islet transplantation may not reach full potential without reliance upon alternative islet sources. Porcine islets are favored for xenotransplantation but mature pigs (>2yrs) present logistic and economic challenges and young pigs (3-6 m) have not yet proved to be an adequate source. Recent findings for the plasticity of immature porcine islets in culture demand a greater understanding of their biology in vitro and in vivo.

Methods: Islets were isolated by conventional collagenase (Roche MTF) digestion of 7 juvenile pancreata (3m; 25kg Yorkshire pigs) and characterized using a battery of tests at various time points during culture in silicone membrane flasks. Islet biology assessment included oxygen utilization, insulin secretion (static and perifusion), histopathology and in vivo function using the diabetic nude-mouse bioassay.

Results: Islet yields (1823±158 IEQ/g) comprised a high proportion (>90%) of small islets (<100µm), and purity after optiprep density gradient purification was 64±6%. Morphologically, islets appeared as “grape-like” clusters on day-0, and loosely disaggregated at day-1. Further culture showed a transition of the tissue to more condensed aggregated structures comprising both exocrine and endocrine cells by day-6. Histopathology confirmed both insulin and glucagon staining of the cultured aggregates and the islet grafts excised after 30days. Nuclear staining (Ki67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 178±20 nmol/min/ng DNA and physiological function was intact by both static and dynamic glucose stimulation. In vivo function was confirmed by sustained (>30d) normalization of blood glucose in (8 of 17) transplants.

Conclusion: Preparation and culture of juvenile porcine islets as a readily available source for islet transplantation requires specialized conditions. These immature islets undergo apparent plasticity in culture involving aggregation with contaminating exocrine tissue to form fully functional “pseudopancreas” structures.

P-202

Biological characterization of isolated juvenile porcine islets as a potential source of tissue for xenotransplantation.

B.P. Weegman¹, M.J. Taylor², S.C. Baicu², R. Pongratz³, K.R. Mueller¹, T.D. O'Brien⁴, G. Cline³, J. Wilson⁵, K.K Papas^1
1 University of Minnesota, Department of Surgery, Minneapolis, MN, USA; ² Cell and Tissue Systems Inc., N. Charleston, SC, USA; ³ Yale University, New Haven, CT, USA; ⁴ University of Minnesota, Department of Veterinary Population Medicine, Saint Paul, MN, USA; ⁵ Wilson Wolf Manufacturing, New Brighton, MN, USA

Objective: Limited availability of donor human pancreata means that clinical islet transplantation may not reach full potential without reliance upon alternative islet sources. Porcine islets are favored for xenotransplantation but mature pigs (>2yrs) present logistic and economic challenges and young pigs (3-6 m) have not yet proved to be an adequate source. Recent findings for the plasticity of immature porcine islets in culture demand a greater understanding of their biology in vitro and in vivo.

Methods: Islets were isolated by conventional collagenase (Roche MTF) digestion of 7 juvenile pancreata (3m; 25kg Yorkshire pigs) and characterized using a battery of tests at various time points during culture in silicone membrane flasks. Islet biology assessment included oxygen utilization, insulin secretion (static and perifusion), histopathology and in vivo function using the diabetic nude-mouse bioassay.

Results: Islet yields (1823±158 IEQ/g) comprised a high proportion (>90%) of small islets (<100µm), and purity after optiprep density gradient purification was 64±6%. Morphologically, islets appeared as “grape-like” clusters on day-0, and loosely disaggregated at day-1. Further culture showed a transition of the tissue to more condensed aggregated structures comprising both exocrine and endocrine cells by day-6. Histopathology confirmed both insulin and glucagon staining of the cultured aggregates and the islet grafts excised after 30days. Nuclear staining (Ki67) confirmed mitotic activity consistent with the observed plasticity of these structures. Metabolic integrity was demonstrated by oxygen consumption rates = 178±20 nmol/min/ng DNA and physiological function was intact by both static and dynamic glucose stimulation. In vivo function was confirmed by sustained (>30d) normalization of blood glucose in (8 of 17) transplants.

Conclusion: Preparation and culture of juvenile porcine islets as a readily available source for islet transplantation requires specialized conditions. These immature islets undergo apparent plasticity in culture involving aggregation with contaminating exocrine tissue to form fully functional “pseudopancreas” structures.