(569i) Bile Acid and Cholesterol Metabolism in 3D Liver Mimics

The liver is responsible for performing many important functions in the body including detoxification, metabolism, and is an integral part of the body's immune system. One particularly important hepatic function is the synthesis of bile acids (BAs) representing the principal route of cholesterol metabolism and clearance from the body. Bile acids are synthesized through a cascade of enzymatic reactions mediated by specific cytochrome P450 enzymes and controlled by intricate feed back interactions between BAs and nuclear receptors. Control of the size and composition of the bile acid pool is tightly regulated allowing for normal hepatic function to be differentiated from the onset of disorders such as cholestasis through BA analysis.

The in vitro study of bile acid synthesis by cultured hypatocytes is limited due to the minute amount of BAs produced in conjunction with the loss of hepatocyte phenotype during culture. We have developed a HPLC/MS technique with a limit of detection below 1 ng/mL, allowing for the simultaneous separation and detection of primary rat BAs. The BAs detected with this technique are unconjugated cholic, chenodeoxycholic (CDCA), and muricholic acid as well as their glycine and taurine conjugates. In addition to studying the BA secretions in hepatocyte monolayer (HM) and collagen sandwich (CS) cultures we profiled BA production by hepatocytes in the novel 3D liver mimic system.

The 3D liver mimic system is designed to reproduce the stratified in vivo liver architecture where the Space of Disse separates hepatocytes from liver sinusoidal endothelial cells (LSECs). The separation and co-culture of primary rat hepatocytes and LSECs in the 3D liver mimic is achieved with layer-by-layer deposition of a porous self-assembled polyelectrolyte scaffold. The scaffold enables cellular interaction through signaling pathways which are responsible for the maintenance of each cell's individual phenotype and hepatic specific functions. Our research has shown undetectable secretion of unconjugated BAs, but both glycocholic and taurocholic acid are synthesized in vitro. At day 3 in both HM and CS cultures CDCA is almost completely metabolized to nearly undetectable concentrations from those observed in fresh culture medium. The same trend continues through day 8 in the CS. However, the metabolic activity in HM cultures decreases from day 3 to day 8 resulting in a 15-fold increase in conjugated CDCA between the two time points. The normal rat BA pool contains muricholic acid, a rat specific BA, which is a 6-β-hydroxylase product of CDCA metoabilism. Therefore, we expect metabolism of CDCA to result in a corresponding increase in muricholic acid if liver specific function is maintained in a hepatocyte culture. We are currently working to verify the 6-β-hydroxylation of CDCA to muricholic acid using a combination of gene expression and proteomic approaches.