Hypothermic storage of periportal and perivenous rat hepatocytes.
High yields of intact parenchymal cells are produced by the two-step Digitonin-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 time-limited use of the isolated cells unless those other methods, such as primary cultures, are employed. An alternative option to have cells ready to be used for several days, is the cold storage in University of Wisconsin solution as a preservation solution. This procedure is easy, not too expensive, and does not require specialized equipment. We study the competence of this system to maintain liver cells: mixed or total cells and cell-enriched fractions. We affirm viability of hepatocytes during hypothermic storage (UW-96 h-4 degrees C) by Trypan Blue exclusion, the capacity to retain cytoplasmic enzymes, metabolic competence to maintain total Glutathione content, and immunocytochemistry (gene detection). After 96 h of cold storage, mixed cells and cell-enriched fractions, were submitted to normothermic incubation (120 min, 37 degrees C) and we check Trypan Blue exclusion, cytoplasmic enzyme release, and the capacity of cell populations to synthesize urea. The results show that it is possible to use, after several days of storage, mixed liver cells and cell-enriched fractions in metabolic and gene expression studies. This procedure allows us to reduce the number of experimental animals needed, to save experimental time and costs, and to facilitate further studies in vitro about the basis and consequences of metabolic heterogeneity of the liver cell plate.
Biología Molecular, Instituto de Fisiología Experimental, Consejo Nacional de Investigaciones Científicas y Técnicas, Rosario, Argentina.
E E
EE
Guibert
M G
MG
Mediavilla
M E
ME
Mamprin
J V
JV
Rodríguez
D000818:Animals; D002469:Cell Separation / Q000379:methods; D002470:Cell Survival; D017364:Collagenases / Q000378:metabolism; D015925:Cryopreservation; D004072:Digitonin / Q000378:metabolism; D007150:Immunohistochemistry; D008099:Liver / Q000166:cytology; D008102:Liver Circulation; D008297:Male; D011169:Portal Vein; D051381:Rats; D017208:Rats, Wistar; D014343:Trypan Blue
D017364:Collagenases; D014343:Trypan Blue; D004072:Digitonin