Maintenance of stimulus-secretion coupling and single beta-cell function in cryopreserved-thawed human islets of Langerhans.
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.
Department of Internal Medicine, Washington University Medical Center, Box 8126, Saint Louis, MO 63110, USA. mislers@msnotes.wustl.edu
Stanley
S
Misler
Adam
A
Dickey
David W
DW
Barnett
D000200:Action Potentials; D002118:Calcium / Q000502:physiology; D015925:Cryopreservation; D004594:Electrophysiology; D005089:Exocytosis; D005934:Glucagon / Q000494:pharmacology; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D050417:Insulin-Secreting Cells / Q000187:drug effects / Q000502:physiology*; D007515:Islets of Langerhans / Q000502:physiology*; D011189:Potassium Chloride / Q000494:pharmacology; D019810:Sodium Azide / Q000494:pharmacology; D013004:Somatostatin / Q000494:pharmacology; D013779:Tetrodotoxin / Q000494:pharmacology
D007328:Insulin; D013779:Tetrodotoxin; D013004:Somatostatin; D011189:Potassium Chloride; D005934:Glucagon; D019810:Sodium Azide; D005947:Glucose; D002118:Calcium