HYPOTHERMIC STORAGE OF PERIPORTAL AND PERIVENOUS RAT HEPATOCYTES

High yields of intact parenchymal cells are produced by the two-step D igitonin-collagenase perfusion of whole liver, and it has gained wide acceptance for biochemical and cellular analyses of zonal hepatocytes, The development reached by this methodology is in contrast to the tim e-limited use of the isolated cells unless those other methods, such a s primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University o f Wisconsin solution as a preservation solution. This procedure is eas y, not too expensive, and does not require specialized equipment. We s tudy the competence of this system to maintain liver cells: mixed or t otal cells and cell-enriched fractions. We affirm viability of hepatoc ytes during hypothermic storage (UW-96h-4 degrees C) by Trypan Blue ex clusion, the capacity to retain cytoplasmic enzymes, metabolic compete nce to maintain total Glutathione content, and immunocytochemistry (ge ne detection). After 96 h of cold storage, mixed cells and cell-enrich ed fractions, were submitted to normothermic incubation (120 min, 37 d egrees C) and we check Trypan Blue exclusion, cytoplasmic enzyme relea se, and the capacity of cell populations to synthesize urea. The resul ts show that it is possible to use, after several days of storage, mix ed liver cells and cell-enriched fractions in metabolic and gene expre ssion studies. This procedure allows us to reduce the number of experi mental animals needed, to save experimental time and costs, and to fac ilitate further studies in vitro about the basis and consequences of m etabolic heterogeneity of the liver cell plate. (C) 1998 Elsevier Scie nce Inc.