Comparative studies on characterization of liver cells isolated from adult rats by trypsin-and collagenase-perfusion

Primary cultures of liver cells isolated from adult rats by trypsin-and collagenaseperfusion technique were carried out to compare biological and biochemical properties of the enzymatically prepared liver cells. (1) The population of isolated liver cells prepared with trypsin was rich in cells with smaller sizes compared with that with collagenase. On the other hand, the population of the collagenase-dispersed liver cells contained many more cells with larger sizes as compared with the former. (2) The cell attachment efficiency on the 1st culture day was about twice as high in the liver cells prepared with collagenase than with trypsin. (3) Mature hepatocytes isolated by collagenase-perfusion could be maintained in primary culture for a longer time than those by trypsin-perfusion. In primary culture of the trypsin-dispersed liver cells, immature hepatocytes started growing after a lag period of 1 day and the number of cultured cells reached a maximal level on the 8th culture day. However, the collagenase-dispersed liver cells in primary culture continued to decrease in number from the 1st to 10th culture day, and then the remaining immature hepatocytes started growing gradually. Furthermore, earlier proliferation of immature hepatocytes could not be induced by trypsinization of the collagenase-dispersed liver cells 1day after initiation of primary culture. (4) Both of the enzymatically prepared liver cells showed albumin production, and exhibited G6Pase and TAT activities for a week in primary culture. The levels of G6Pase activities were nearly the same in both of the enzymatically prepared liver cells. But albumin production was higher in the liver cells prepared with collagenase than with trypsin. Especially, the level of TAT activities up to 2nd culture day was about three times higher in the liver cells prepared with collagenase than those with trypsin. (5) In both of the primary culture systems, combined supplementation of dexamethasone (10-5M) and insulin (10μg/ml) consistently improved the cell attachment efficiency and was also very effective for maintenance of mature hepatocytes. Furthermore, these hormones enhanced albumin production and TAT activities in both of the enzymatically prepared liver cells in primary culture. But the liver cells prepared by collagenase-perfusion had a higher response to these hormones than those by trypsinperfusion. (6) From these observations described above, it seems that the population of liver cells prepared with trypsin would contain much more immature hepatocytes with a higher proliferative ability than that with collagenase. On the other hand, collagenase would isolate highly intact mature hepatocytes from rat liver as compared with trypsin. Therefore, the use of liver cells isolated by trypsin-perfusion may be desirable for obtaining rapid growth of immature hepatocytes. On the other hand, the use of collagenase dispersed liver cells may be preferred for maintaining mature hepatocytes with specialized liver functions for a longer time in primary culture