Cell culture models preparative techniques

In this chapter more detailed information on the double-cell voltage-clamp setup and protocols for assessing gap junctional conductivity is given, as well as a description of the cell-isolation procedure for this purpose and cell culture models. Information on immunocytochemical localization of gap junctions and on the experimental procedure of preparing specimens and slides for immunohistology is given. A protocol for isolation of gap junction proteins is also outlined. Readers interested in more details of the cell-culture technique regarding incubators, sterile technique, etc., and different isolation and culture protocols are referred to more specialized literature [Lindl and Bauer, 1994 Piper, 1990]. 

To study the in vitro behaviour of carrier molecules, cells may be derived from a cell line or from primary cultures of rat or human liver cells. In general, the latter cells better reflect the in vivo situation than the immortalized cells in a cell line culture. It should be noted, however, that during the isolation procedure the enzymes collagenase and pronase may destroy or damage the target receptors. In vitro preparations that approach the in vivo situation best are liver slices, in which the physiological context of the liver is maintained [229], and the model of the isolated perfused rat liver (IPRL) [230, 231]. These techniques allow us to study carrier-cell interactions within the organ, that is, in the presence of all other liver cells and their secreted mediators. 

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