Fractionation of Hepatic Nonparenchymal Cells

The majority of parenchymal cells from mammalian liver cells can be removed by very low speed centrifugation (50 g) but a simple low-density barrier (1.096 g/ml) is required to remove the remaining parenchymal cells from the 50-g supernatant which contains all of the lower density nonparenchymal cells. Continuous gradients of Nycodenz can provide satisfactory resolution of Kupffer, stellate, and endothelial cells on an analytical basis but the separation of different cell types is not sufficient preparatively. Flotation through a low-density iodixanol barrier can, however, provide a satisfactory enrichment of the least dense nonparenchymal cell – the stellate cells.


INTRODUCTION
The fractionation of hepatic nonparenchymal (sinusoidal) cells (Kupffer cells, stellate cells, endothelial cells, etc.) on continuous metrizamide [1] and Nycodenz gradients [2] has shown that the overlap in banding densities of these cells makes these gradients generally unsatisfactory as a stand-alone procedure to isolate the various cell populations.
Discontinuous gradients of iodinated media are widely used to provide a means of removing residual erythrocytes and parenchymal cell debris from nonparenchymal cell preparations and to prepare a stellate cell-rich fraction.

Preparation of Nonparenchymal Cells
Parenchymal cells are routinely prepared by collagenase digestion of the liver using a tissue perfusion system. These cells are then separated from the nonparenchymal cells by differential pelleting at 50 g for 1-4 min. Although the nonparenchymal cells can be isolated from the 50-g supernatant, the yields are usually low. The most widely used procedure is to perfuse the liver with a mixture of collagenase and Pronase or endotoxin to destroy the parenchymal cells selectively (see Refs. 1 and 2 for details).

Stellate Cells
These are the least dense of the nonparenchymal cells and can be floated effectively away from the rest of the cells. The low-density fraction may contain almost 80% stellate cells [2]. The actual density of the low-density layer used by different workers varies somewhat and some of these variations are presented in the Notes section The following methods are adapted from Bøyum et al. [1] and from Brouwer et a.l [2]. The advantage of using OptiPrep rather than Nycodenz is that the density solutions can be made up by dilution of OptiPrep directly with Gey's Balanced Salt Solution (GBSS), while Nycodenz must be diluted with GBSS minus the NaCl to keep the osmolality below 300 mOsm. 2. Add WS to the cell suspension so that the final concentration of iodixanol is 17% (w/v) iodixanol solution (ρ = 1.096 g/ml). 3. Mix thoroughly but gently. 4. Layer approx. 2 ml of GBSS on top and centrifuge at 400 g for 15 min. at 20°C. 5. Allow the rotor to decelerate without the brake. 6. Collect the cells, which band at the interface between the GBSS and the 17% iodixanol.

STELLATE CELLS
1. Add WS to the cell suspension as described is steps 1-3 above. 2. Dilute W with GBSS to produce a solution containing 11.5% (w/v) iodixanol (see Note 1). 3. Layer 5 ml of this solution over the same volume of cell suspension (in 17% iodixanol); then layer 2 ml of GBSS on top (see Note 2). 4. Centrifuge at 1400 g for 17 min. at 20°C; allow the rotor to decelerate without the brake. 5. Collect the cells, which band at the interface between the GBSS and the 11.5% iodixanol (see Fig. 1).

NOTES
1. This concentration of iodinated density gradient medium is equivalent to approx 1.067 g/ml and has been used both with Nycodenz [1,2] and iodixanol [3,4]. A lower-density concentration (1.053 g/ml) was used by Cassiman et al. [5]. This is equivalent to approx. 9% (w/v) iodixanol. 2. In the method as described by Brouwer et al. [2], the cell suspension was placed in the top layer (ρ = 1.067 g/ml) rather than the bottom layer (ρ = 1.096 g/ml) and this is an alternative strategy.