Preparation and Primary Culture of Liver Cells Isolated from Adult Rats by Dispase Perfusion

The dispase perfusion technique was used to isolate liver cells from adult rats. The optimum conditions for obtaining many isolated liver cells with high viability were an enzyme concentration of 2000 U/ml, a pH of 7.5 and a perfusion time of 20 min. The population of isolated liver cells prepared with dispase consisted of 43.6% cells with diameters less than 20 micron and 56.4% cells with diameters above 20 micron. The isolated liver cells were cultured in basal culture medium either supplemented with or without dexamethasone (1 X 10(-5)M) and insulin (10 micrograms/ml). The addition of hormones to the culture medium improved the attachment efficiency of the isolated liver cells and delayed the disappearance of mature hepatocytes. Epithelial-like clear cells proliferated early in primary culture even in the presence of hormones. Therefore, functioning mature hepatocytes and proliferating epithelial-like clear cells coexisted well in the hormone-containing medium. Furthermore, the number of cultured cells reached a maximal level earlier in the presence of hormones than in the absence of hormones. The level of TAT activity in primary cultured cells was higher up to 3 days after inoculation in the presence of hormones than in their absence. No difference between G6Pase activity in primary cultured cells in the presence of hormones and that in the absence of hormones was found.