Question

Protocol for isolation of liver parenchyma cells

Answer 1

Explanation:

A technique is described for isolation of adult rat hepatocytes from micronodular cirrhotic livers based on a collagenase digestion procedure. Hepatocytes from normal livers and those chronically injured by thioacetamide did not differ with respect to the viability measured by the trypan blue exclusion test or to the cellular concentrations of protein and glycogen, but the triglycerde content of cells from cirrhotic livers was significantly reduced. Hepatocytes isolated from cirrhotic livers are ultrastructurally in a good state of preservation but they appear to be poorer than controls in RER membranes, although the well‐preserved mitochondria are somewhat richer in cristae. No differences were detected between the cell preparations in rates of gluconeogenesis and total de novo fatty acid synthesis, but the secreation of newly synthesized fatty acids was significantly reduced in cells from cirrhotic livers.

Answer 2

$Answer$

• Centrifuge the collected supernatant (step 4.5 and 4.6) at 72 x g, 5 min, 4 °C to eliminate the remaining erythrocytes and hepatocytes.

• Pool both pellets and re-suspend them in HBSS.