Purification of hepatic non-parenchymal cells on a density barrier
Parenchymal and non-parenchymal cells (PC and NPC) may be prepared by collagenase digestion of the liver using a tissue perfusion system. The PC are then separated from the NPC by differential pelleting at 50 g for 1-4 min. It is however necessary to repeat this centrifugation (maybe twice more) to remove PC from the supernatant; moreover the NPC yield is usually low.
It is both more common and more effective to carry out the 50 g centrifugation once; to harvest all the cells from the supernatant by centrifugation at a higher g-force and then use a density barrier prepared from one of the of iodinated density gradient media to resolve the two types of cell.
Many workers prefer a modified perfusion strategy, which involves a mixture of collagenase and Pronase or Clostridium perfringens enterotoxin to destroy the PC selectively [1,2].
The purification of NPC is a starting point for the isolation of Kupffer and endothelial cells; the NPC are then processed further by centrifugal elutriation to obtain reasonably pure populations of these important cells, although there are instances of the reverse situation in which the elutriation is the first step (e.g. ref. 3).
Although metrizamide was widely used for the density barrier step prior to 1984,
Nycodenz subsequently became more popular (over 200 papers) because of its lower toxicity to cells.
More recently iodixanol gradients have become a popular choice and because of the ease of gradient
solution preparation from OptiPrep, the following
methodology is based solely on use of this medium.