Measurement of P450 Difference Spectra Using Intact Cells

Abstract

In vitro models, based on liver cells or tissues, are indispensable in the early preclinical phase of drug development. An important breakthrough in establishing cell models has been the successful high-yield preparation of intact hepatocytes. In this chapter, the practical aspects of the two-step collagenase perfusion method, modified from the original procedure of Seglen, are outlined. Although applicable to the liver of various species, including human, the practical aspects of the method are explained here for rat liver. Critical parameters for the successful isolation of primary rat hepatocytes are highlighted and a troubleshooting guide is provided. In addition, a new development based on the inhibition of histone deacetylase activity is presented. This approach allows inhibition of cell-cycle reentry during hepatocyte isolation, a process known to underlie the dedifferentiation process of cultured hepatocytes.