Perifusion Culture of Hepatocytes

Cultivation of cells in Petri dishes or culture flasks is usually performed by changing the medium after certain periods of time, e.g., 24 h (stationary cultivation). Clearly, the concentration of nutrients and other components as well as of added hormones will decrease over this period because of the metabolic activity of the cultured cells Conversely, metabolic end products such as lactate, ammonium ions and even bile salts will accumulate. In order to replace the sequential changes of the culture medium by a continuous supply, several systems for perifusion or circumfusion of the cells with medium have been designed. Usage of one such approach, reported for cultivation of hepatocytes (1 ), indicated that perifusion was beneficial to the cultured hepatocytes, resulting in a prolonged life-span and an enhanced metabolic performance In particular, it was found that the hormonal response to dexamethasone and glucagon was increased (2 ), partly because steady-state concentrations of hormones are established even at low initial concentrations. Since these early studies, the technique for perifusion has improved and it has been found that oxygen tension within the culture medium is a very critical parameter; surprismgly, in contrast to previous assumptions that oxygen supply in normal stationary cultures is insufficient (3 ), higher oxygen tensions, in conjunction with elevated flow rates, proved to be cytotoxic under perifusion conditions. If this and other special features and requirements are considered, perifusion cultivation has considerable advantages over conventional culture techniques, particularly for studies on biotransformation and drug toxicity (4 , 5 ).