Use of Percoll density gradient centrifugation for preparing isolated rat hepatocytes having long-term viability
✍ Scribed by Christian Dalet; Max Fehlmann; Pascale Debey
- Publisher
- Elsevier Science
- Year
- 1982
- Tongue
- English
- Weight
- 345 KB
- Volume
- 122
- Category
- Article
- ISSN
- 0003-2697
No coin nor oath required. For personal study only.
✦ Synopsis
A method for preparing suspensions of hepatocytes with long-term viability is described. Its originality lies in the use, after conventional preparation, of a Percoll density gradient centrifugation which allows a complete and rapid separation of cell debris and released proteases from the intact hepatocytes. A series of functional reactions characterizing drug metabolism (P-450 level, ethoxycoumarin hydroxylation, methylumbelliferone conjugation) and membrane transport and integrity (c*-aminoisobutyric acid transport and its stimulation by both insulin and glucagon) were conducted, in parallel to the trypan blue exclusion test, to evaluate the metabolic activity of both conventional and Percoll preparations. This investigation clearly demonstrates that the use of Percoll density gradient centrifugation significantly increases (from 5-6 to 30-35 h) the half-life of freshly isolated hepatocyte suspensions.
' Abbreviations used: BSA, bovine serum albumin; AIB, a-amino-[ 1 -'%]isobutyric acid; KRb, Krebs-Ringer bicarbonate buffer; Hepes, 4-(2-hydroxyethyl) I-piperazineethanesulfonic acid.