title: Comparison of top and bottom loading of a dextran gradient for rat pancreatic islet purification.
abstract: Rat pancreatic islet yields obtained with dextran gradient purification were compared after suspending the digest into either the top or the bottom layer of the gradient. A 5-layer discontinuous gradient was used, which consisted of 16 ml 31% dextran as bottom layer, overlayered with 25%, 23%, 20% and 11% dextran (4 ml each). When the digest of 1 rat pancreas was suspended into the top layer of the gradient, the total number of islets obtained from the 11-20, 20-23 and 23-25% interfaces was 862 +/- 38, 240 +/- 39 and 54 +/- 5, respectively. From this gradient, also 1409 +/- 81 islets were retrieved from the bottom layer (i.e., exocrine pellet). In contrast, when the pancreas digest was suspended into the bottom layer of the gradient, 1964 +/- 63, 435 +/- 42, and 177 +/- 34 islets were obtained from the successive interfaces, and only 50 +/- 20 islets from the exocrine pellet. The total islet volume obtained from the two uppermost interfaces was 3.46 +/- 0.31 microliters after top-loading, and 4.93 +/- 0.16 microliters after bottom-loading (n = 7, p < 0.01). When the islets retrieved from one bottom loaded gradient were transplanted into either 1 (n = 6) or 2 (n = 9) diabetic recipients, glucose levels normalized in all instances. We therefore conclude that a bottom-loaded dextran gradient separates islets from exocrine tissue effectively, resulting in significantly higher islet yields than obtained with a top-loaded dextran gradient.
authors.affiliation: Department of Surgery, University of Groningen, The Netherlands.
authors.forename: W M
authors.initials: WM
authors.lastname: Fritschy
authors.forename: P T
authors.initials: PT
authors.lastname: van Suylichem
authors.forename: G H
authors.initials: GH
authors.lastname: Wolters
authors.forename: R
authors.initials: R
authors.lastname: van Schilfgaarde
mesh_terms: D000818:Animals; D001786:Blood Glucose / Q000378:metabolism; D002469:Cell Separation / Q000295:instrumentation / Q000379:methods; D002470:Cell Survival; D002501:Centrifugation, Zonal / Q000295:instrumentation / Q000379:methods; D003911:Dextrans; D003921:Diabetes Mellitus, Experimental / Q000097:blood / Q000601:surgery*; D007515:Islets of Langerhans / Q000166:cytology*; D016381:Islets of Langerhans Transplantation / Q000502:physiology*; D051381:Rats
chemical_list: D001786:Blood Glucose; D003911:Dextrans