CRYOGENIC STORAGE OF ISOLATED, PURIFIED PORCINE PANCREATIC ISLETS

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Abstract

Highly purified islets of Langerhans were prepared in the present study from adult pigs by collagenase digestion and density gradient purification. After overnight culture, the tissue was equilibrated with DMSO at 25°C, supercooled to −7.5°C, nucleated, slowly cooled at 0.25°C/min to −40°C, and stored at −130°C. Then, after variable periods of storage, the is-lets were rapidly thawed at 37°C. Postthaw actual islet and islet equivalent (150-µm sized islets) recovery were 75±7% and 66±4%, respectively. The frozenthawed porcine islets maintained good morphology on histological staining by hematoxylin-eosin and aldehyde-fuchsin. Upon perifusion, basal insulin secretion was 43±10 and 67±18 pmol/L from noncryopreserved, control islets, and cryopreserved islets, respectively (P=0.2). Peak insulin release at 16.7 mmol/L glucose was 85±28 pmol/L from noncryopreserved islets and 157±48 pmol/L from the frozen-thawed islets (P=0.1). When 10 mmol/L theophylline was added to 16.7 mmol/L glucose, the secretion of the hormone peaked to 221±83 (control islets) and 479±140 pmol/L (cryopreserved islets, P=0.1). Total insulin secretion differed significantly for the noncryopreserved and the cryopreserved islets at both 16.7 mmol/L (1412±306 vs. 3756±764 pmol/L, respectively, P=0.007) and 16.7 mmol/L glucose plus 10 mmol/L theophylline (2161± 371 vs. 7505±2075 pmol/L, respectively, P=0.011). Normoglycemia was restored within 7 days from implantation in temporarily immunosuppressed (aL3T4 antibody) mice with streptozotocin-induced diabetes by transplanting 1500–2000 cryopreserved porcine islets under the kidney capsule. Mean survival time of frozen-thawed islet xenografts (39±3 days) was similar to that of noncryopreserved islet xenografts (43±6 days). This study demonstrates that cryogenic storage is feasible of isolated porcine islets, with the frozenthawed pancreatic endocrine tissue maintaining morphological integrity and both in vitro and in vivo viability. Further studies are needed to define the effect of cryopreservation on the immunogenic properties of porcine islets.

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