Journal
PFLUGERS ARCHIV-EUROPEAN JOURNAL OF PHYSIOLOGY
Volume 450, Issue 6, Pages 395-404Publisher
SPRINGER HEIDELBERG
DOI: 10.1007/s00424-005-1401-y
Keywords
human islets of Langerhans; cryopreservation; beta-cells; insulin; secretion; stimulus-secretion coupling; electrophysiology; depolarization-secretion coupling; exocytosis
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Funding
- NIDDK NIH HHS [DK37380] Funding Source: Medline
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Studies of stimulus-secretion coupling in human beta-cells have been hampered by poor availability of tissue due to variability of the supply of cadaver pancreati and in the adequacy of enzymatic liberation of islets as well as by the shunting of isolates into transplant trials. Here we establish that aliquots of islets, several from high-quality but low-yield islet isolates (50,000-100,000 islets), cryopreserved and then thawed as needed, respond to glucose in a calcium- and metabolic-dependent fashion. Insulin secretion is modulated by blockers of voltage-dependent Na+ and Ca2+ channels, and paracrine hormones (glucagon and somatostatin) in manners indistinguishable from fresh tissue preparations. Using single-cell electrophysiological and electrochemical assays we demonstrate that single beta-cells from cryopreserved islets display (1) stimulus-depolarization coupling based on rapid closure of K+ (ATP) channels; (2) action potential electrogenesis with upstrokes based on voltage-dependent Na and Ca currents; and (3) Ca2+ entry-mediated depolarization-exocytosis coupling sustained over multiple bouts of stimulation and modulated by paracrine hormones. All of these features are indistinguishable from those seen in single cells from freshly harvested islets. These results support the utility of cryopreservation, even of low-yield but functional isolates, as a means of ensuring a steady source of repeatedly accessible tissue for research on normal and diabetic islets.
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