Pancreatic islet purification using bovine serum albumin: the importance of density gradient temperature and osmolality.
Euro-Ficoll and bovine serum albumin (BSA) are two of the most commonly used density gradient media for the purification of pancreatic islets. Euro-Ficoll is based upon Euro-Collins, a cold storage medium, and must, therefore, be used at 4 degrees C. The ionic composition of BSA, however, is likely to contribute to hypothermic cellular swelling, and this may influence the efficiency of islet purification using this medium at 4 degrees C. Experience in this laboratory also suggested that batch-to-batch variation in islet purity using BSA was related to differences in BSA osmolality. The aim of this study was to assess the effect of gradient medium temperature and osmolality on the purification of human and porcine islets using BSA. Pancreata were collagenase-digested, and islets were purified on continuous linear density gradients of BSA. The distribution of insulin and amylase in each gradient was assayed, and used to calculate the median density of islets and exocrine tissue, and the efficiency of islet purification (% amylase contamination at a fixed insulin yield), using: 1) gradient osmolalities of 300, 400, and 500 mOsm/kg H2O (seven porcine pancreata), and 2) gradients at 4 degrees C and at 22 degrees C (eight human and seven porcine pancreata). Increase in density gradient osmolality produced increases in porcine exocrine tissue density which exceeded changes in islet density, resulting in improved islet purity, maximal at a BSA osmolality of 400 mOsm/kg H2O. For human pancreata there was no significant change in pancreatic tissue densities nor islet purity with temperature.(ABSTRACT TRUNCATED AT 400 WORDS)
Chadwick
D R
DR
Department of Surgery, Leicester Royal Infirmary, UK.
Robertson
G S
GS
Toomey
P
P
Contractor
H
H
Rose
S
S
James
R F
RF
Bell
P R
PR
London
N J
NJ
D000293:Adolescent; D000328:Adult; D000368:Aged; D000681:Amylases / Q000378:metabolism; D000818:Animals; D002452:Cell Count; D002469:Cell Separation / Q000379:methods*; D002500:Centrifugation, Isopycnic / Q000379:methods; D006801:Humans; D007515:Islets of Langerhans / Q000166:cytology* / Q000201:enzymology; D016381:Islets of Langerhans Transplantation; D008875:Middle Aged; D009994:Osmolar Concentration; D010179:Pancreas / Q000166:cytology / Q000201:enzymology; D012710:Serum Albumin, Bovine; D013552:Swine; D013696:Temperature
D012710:Serum Albumin, Bovine; D000681:Amylases