Dynamic perifusion to maintain and assess isolated pancreatic islets.
Advances in human islet transplant techniques are hampered by the inability to assess the quality of isolated islets. A flow culture system was developed to perifuse isolated pancreatic islets or cultured beta-cell lines in order to continuously and noninvasively assess cell function and viability with high kinetic resolution. Continuous perifusion of large amounts of islet tissue as isolated from human pancreata enables the use of noninvasive measurement technologies not previously applied to islets. To compare dynamic perifusion of tissue at high density with conventional static cultures, we measured glucose-stimulated insulin secretion and O2 consumption of large amounts of INS-1 cells (45-65 x 10(6)) to confirm that perifused cells were adequately supplied with oxygen and nutrients and remained functionally responsive. Isolated human and monkey islets that were perifused for 18 h showed robust biphasic insulin secretion in response to a step increase in glucose, demonstrating the ability to maintain islets and the high kinetic resolution of the system. As an example of the system's ability to resolve multiple indicator dilution experiments, the retention of [3H]-glibenclamide was kinetically distinguished from that of an extracellular marker. In summary, the perifusion system is able to maintain healthy cells, assess insulin secretion and metabolite fluxes such as oxygen consumption and lactate production, and characterize the kinetics of the interaction between radiopharmaceuticals and islet cells. The ability to systematically assess the metabolic and functional viability of islets will facilitate the optimization of islet isolation procedures, islet transplantation studies, and islet storage methodologies.
Sweet
Ian R
IR
Department of Medicine, University of Washington, Seattle 98195, USA. isweet@u.washington.edu
Cook
Daniel L
DL
Wiseman
Robert W
RW
Greenbaum
Carla J
CJ
Lernmark
Ake
A
Matsumoto
Shinichi
S
Teague
Jeanette C
JC
Krohn
Kenneth A
KA
D000818:Animals; D018929:Cell Culture Techniques / Q000295:instrumentation / Q000379:methods; D002460:Cell Line; D002478:Cells, Cultured; D005947:Glucose / Q000494:pharmacology; D006801:Humans; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000166:cytology* / Q000187:drug effects / Q000378:metabolism / Q000502:physiology*; D007700:Kinetics; D008254:Macaca nemestrina; D010101:Oxygen Consumption; D010477:Perfusion / Q000379:methods; D013997:Time Factors
D007328:Insulin; D005947:Glucose