Non-enzymatic isolation and culture of channel catfish hepatocytes

An alternative method has been developed for isolating and culturing hepatocytes from livers of channel catfish. Hepatocytes are prepared using a collagenase-free perfusion system that relies on the chelating properties of ethylenediamine tetraacetic acid (EDTA). Hepatocyte yields of up to 3.6 x 10(8) cells per 100 g body weight have been achieved with initial viabilities routinely exceeding 95%. Cells isolated by this method and incubated in osmotically corrected culture medium at physiological pH have been maintained for several weeks in culture with minimal cell loss. During the first 24-48 h of culture, hepatocytes begin to link together and show structures that closely resemble those seen in intact liver (e.g. bile canaliculi, sinusoids). Cells cultured at 15 degrees C for 7 days maintain levels of glucose-6-phosphate dehydrogenase (G6PDH), 6-phosphogluconate dehydrogenase (6PGDH), and lactate dehydrogenase (LDH), activity similar to those measured in vivo.