Mass isolation of highly purified canine islets using an automated method and Histopaque Ficoll gradients.
Eighteen pancreata from adult mongrel dogs were used for the study of islet isolation. The pancreas was distended with collagenase in Hanks' solution. The automated screen method and Histopaque Ficoll gradients were used to isolate and purify the canine islets. In vitro, the viability of isolated islets was assessed by both histology and perifusion studies. In vivo, the islet function was evaluated by using a nude mice xenograft model. Fair to good isolation and purification was found in 12 experiments. Before and after purification, the isolated islet count was 4767.1 +/- 560.1 and 3637.7 +/- 333.4 islet equivalence (I.E.)/gm pancreatic tissue. The purity was above 90%. Aldehyde Fuchsin stain disclosed islets with copious beta granules. The stimulation index of islets responding to 16.7 mM glucose plus 1 mM 3-isobutyl-1-methylxanthine (IBMX) versus 1.67 mM glucose was 12.93 +/- 4.75. Normoglycemia was restored and maintained for up to 2 weeks in 7 of 10 and up to 3 weeks in 5 of 10 diabetic nude mice transplanted with canine islets. In conclusion, the automated screen method and Histopaque Ficoll gradients afford a good yield of highly purified canine islets, and functional viability was verified both in vitro and in vivo. This will be an ideal model for isolation of human islets.
Chern
H T
HT
Department of Surgery, Tri-Service General Hospital, National Defense Medical Center, Taipei, Taiwan, R.O.C.
Chen
C F
CF
Leu
F J
FJ
Tsou
S S
SS
Chang
T M
TM
Sun
A M
AM
D000818:Animals; D001331:Automation; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods*; D002499:Centrifugation, Density Gradient; D003973:Diatrizoate; D004285:Dogs; D004867:Equipment Design; D005260:Female; D005362:Ficoll; D006943:Hyperglycemia / Q000601:surgery; D007003:Hypoglycemia / Q000209:etiology; D007328:Insulin / Q000378:metabolism; D000078790:Insulin Secretion; D007515:Islets of Langerhans / Q000378:metabolism; D016381:Islets of Langerhans Transplantation / Q000009:adverse effects / Q000502:physiology; D008297:Male; D051379:Mice; D008819:Mice, Nude; D010179:Pancreas / Q000166:cytology*
D007328:Insulin; D003973:Diatrizoate; D005362:Ficoll; C053816:histopaque