Control of the growth and differentiation of cultured hepatocytes is a critical component of the use of these living cells for in vitro toxicology and bioartificial liver assist devices. The phenotype of cultured hepatocytes has been manipulated via the substrate chemistry, morphology and compliance and also by altering the levels of nutrients, hormones and growth factors. A class of compounds that may also affect the phenotype of cultured hepatocytes is differentiation agents, a number of which are used in therapeutic or preventive settings for cancer. Retinoic acid is a metabolite of vitamin A that acts as a ligand on a set of cytoplasmic receptors that translocate to the nucleus and are trans-acting agents affecting transcription of target genes. We have found that retinoic acid isoforms, particularly the naturally occurring all-trans retinoic acid (tRA), exert strong effects on the behavior of both human hepatoma (HepG2) cells and on primary rat hepatocytes.

At high levels, retinoic acid can induce apoptotic cell death in a variety of transformed cell lines, including hepatoma cells such as HepG2. By reducing the dose by several orders of magnitude, we found that we can inhibit the growth of HepG2 cells with tRA while largely avoiding cell death, maintaining a viable population. Moreover, this population exhibits significantly higher albumin and urea productivity as compared to cells cultured in the absence of tRA. Thus, hepatoma cell cultures can be switched from a proliferative to a differentiated phenotype by the addition of tRA.

We also observe that tRA affects the differentiation status of cultured primary rat hepatocytes. A markedly more aggregated phenotype is observed in hepatocytes cultured in the presence of tRA as compared to those without. This effect was highly dependent on the time of tRA induction being within the first two days of culture. Experiments conducted in conventional hepatocyte medium containing supraphysiological levels of insulin showed an increase in urea output by hepatocytes but depressed albumin output. On the other hand, when insulin was decreased to physiological levels, hepatocytes cultured in tRA exhibited increased levels of albumin accompanied by somewhat depressed levels of urea output. This suggests that combination of hormones (insulin and retinoic acid) can be used to tune hepatic metabolism. We also observed that hepatocytes cultured in tRA tend to accumulate more lipidic compounds, as detected by Oil Red O staining. Since others have found a negative correlation between intracellular triglycerides and hepatic differentiated function, future work will be directed towards enhancing the beneficial effects of tRA by inhibiting the uptake and storage of these species.