Enrichment of hepatic Kupffer cells in a discontinuous gradient

Parenchymal and non-parenchymal cells (PC and NPC) may be prepared by collagenase digestion of the liver using a tissue perfusion system. The PCs 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; it uses a mixture of collagenase and Pronase or Clostridium perfringens enterotoxin to destroy the PC selectively [1,2]. Hendriks et al [3] preferred Pronase because of the uncertain commercial availability of the enterotoxin and the latter’s possible cause of cell blebs. 

One- or two-layer density gradient centrifugation alone may not be sufficiently discriminating to provide a pure preparation of Kupffer cells, but this technique can provide an important initial enrichment for these cells prior to the use of centrifugal elutriation, adherence of the Kupffer cells to a plastic surface; sometimes both elutriation and surface adherence are used. 

Antibody-bound magnetic beads have also been used as a final purification step. See Section 5 for more information about additional procedures. The methods in this Application Sheet may simply provide a pure preparation of total NPC or of a NPC fraction also impoverished in the lighter stellate cells (see Section 4)