Hepatocyte isolation from pig livers after warm ischaemic injury

Abstract Hepatocyte cultures have been used extensively for a wide variety of physiological, pharmacological and experimental studies. The warm ischaemic period before isolation is kept to a minimum to achieve a high yield of cells isolated and a good viability for culture. We have recently introduced a new concept of liver resuscitation after warm ischaemia that is based on a 3-h reperfusion period with an improved perfusate and simultaneous dialysis. In this study, we applied the new technique for hepatocyte isolation from livers subjected to 80 min of complete ischaemia at 37 °C. Cell yield was improved by a resuscitating perfusion from 58% to 73% and viability from 39% to 76%.     

Growth inhibition and transformation of a human fetal tracheal epithelial cell line by long-term exposure to diethylnitrosamine

In order to obtain more information on the in vitro transformationof human cells, a human fetal tracheal epithelial cell line(FHET16/5) was exposed for a long time to diethylnitrosamine(DEN). In 20 passages, this cell line (diploid, male) maintainedstrong immunohistochemical reactivity for carcino-embryonnicantigen and wool merokeratin; it was negative for vimentin.The cells contained PAS-positive mucous substances and ultrastructurallywere found to have desmosomelike attachments. Treatment of thecells was with 0.3% dimethyl sulphoxide (DMSO), or DMSO with150, 450, 1000 or 2000 µg/ml of DEN. It was started atthe ninth passage and continued for six passages over 9 weeksfor the control (DMSO) and the three lowest control doses ofDEN, and for three passages over 9 weeks for the 2000 µg/mlDEN group. Cells grown for 13 days after the end of treatmentwere plated in soft agar and injected subcutaneously in nudemice. The frequency of anchorage-independent colonies grownin soft agar was directly related to DEN dose. Colony-formingefficiency, as an expression of toxic effect, was also dosedependent. Autoradiographically detected unscheduled DNA synthesisindicated an association between anchorage-independent transformationand DNA alterations induced by DEN. Cells injected into nudemice did not produce tumours during a 6-month period, but invasivenesswas observed when cells from the 2000 µg/ml DEN groupwere transplanted on the dermis of cultured chick embryo skin.The results indicate that DEN causes anchorage-independent transformationaccompanied by unscheduled DNA synthesis in a fetal human trachealepithelial cell line.