| Popis: |
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, p0.01). 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. |
