Engineered Three-Dimensional Liver Mimics Recapitulate Critical Rat-Specific Bile Acid Pathways

Abstract

A critical hepatic function is the maintenance of optimal bile acid (BA) compositions to achieve cholesterol homeostasis. BAs are rarely quantified to assess hepatic phenotype in vitro since existing analytical techniques have inadequate resolution. We report a detailed investigation into the biosynthesis and homeostasis of eight primary rat BAs in conventional in vitro hepatocyte cultures and in an engineered liver mimic. The threedimensional (3D) liver mimic was assembled with layers of primary rat hepatocytes and liver sinusoidal endothelial cells. A high-pressure liquid chromatography and mass spectrometry technique was developed with a detection limit of 1 ng/mL for each BA, which is significantly lower than previous approaches. Over a 2-week culture, only 3D liver mimics exhibited the ratio of conjugated cholic acid to chenodeoxycholic acid that has been observed in vivo. This ratio, an important marker of BA homeostasis, was significantly higher in stable collagen sandwich cultures indicating significant deviation from physiological behavior. The biosynthesis of taurob-muricholic acid, a key primary rat BA, doubled only in the engineered liver mimics while decreasing in the other systems. These trends demonstrate that the 3D liver mimics provide a unique platform to study hepatic metabolism.

Optimized protocol for the hepatic differentiation of induced pluripotent stem cells in a fluidic microenvironment

“…However, no specific production of GCDCA was observed in both Petri Dishes and biochips, while TCDCA was found to be depleted in the later. In regard to TCDCA, this measurement could be correlated to previous reports in in vitro rat cultures for which the concentration of TCDCA is found to be decreasing as the phenotypes of hepatocytes is preserved (Detzel, Kim & Rajagopalan, 2010). These results show that in the differentiation and especially in biochips some of the pathways of the liver are being activated.…”

“…In regard to TCDCA, this measurement could be correlated to previous reports in in vitro rat cultures for which the concentration of TCDCA is found to be decreasing as the phenotypes of hepatocytes is preserved (Detzel, Kim & Rajagopalan, 2010). Measurements of bile acids indicated that in Petri dishes and biochips, CA and CDCA were converted.…”

Bioreactor technologies to support liver function in vitro

“…This is a promising step toward creating an independently accessible biliary network, though the PDMS might prove highly absorptive to many compounds solubilized in bile. Other tissue engineering approaches that have not yet been adapted to microfluidic culture but that enhance biliary function as assessed by production of biliary components may provide additional ways to manipulate cells in microfluidic culture [159]. Further applications or commercial fate of this Corning Incorporated device are as yet unpublished.…”

Transcriptomic Analysis of Hepatic Cells in Multicellular Organotypic Liver Models

“…2a ). As in the case of cholesterol biosynthesis, we compared the expression levels of these genes in LSECs to hepatocytes from the 3DHLK model, since only hepatocytes have been shown to produce BAs 58 , 74 , 75 . Using GSEA, we found no enrichment of gene sets related to BA biosynthesis ( q -value > 0.01), suggesting similar expression levels among these genes in both LSECs and hepatocytes and supporting the possibility of bile acid biosynthesis in LSECs.…”

“…Bile acid biosynthesis is a well known function of the liver 58 . We have previously shown that 3DHL models synthesize and metabolize bile acids 74 . Here, we find transcriptional support for altered bile acid metabolism in LSECs cultured in the organotypic liver models.…”