Utility of human hepatocyte spheroids without feeder cells for evaluation of hepatotoxicity.

We investigated the utility of three-dimensionally cultured hepatocytes (spheroids) without feeder cells (Sph(f-)) for the prediction of drug-induced liver injury (DILI) in humans. Sph(f-) and spheroids cultured on feeder cells (Sph(f+)) were exposed to the hepatotoxic drugs flutamide, diclofenac, isoniazid and chlorpromazine at various concentrations for 14 days, and albumin secretion and cumulative leakages of toxicity marker enzymes, aspartate aminotransferase (AST), alanine aminotransferase (ALT), lactate dehydrogenase (LDH) and γ-glutamyl transpeptidase (γ-GTP), were measured. The cumulative AST, LDH or γ-GTP leakages from Sph(f-) were similar to or greater than those from Sph(f+) for all drugs tested, although ALT leakages showed no consistent difference between Sph(f+) and Sph(f-).

Preliminary Evaluation of Three-Dimensional Primary Human Hepatocyte Culture System for Assay of Drug-Metabolizing Enzyme-Inducing Potential.

Drug-induced liver injury (DILI) is a common reason for withdrawal of candidate drugs from clinical trials, or of approved drugs from the market. DILI may be induced not only by intact parental drugs, but also by metabolites or intermediates, and therefore should be evaluated in the enzyme-induced state. Here, we present a protocol for assay of drug-metabolizing enzyme-inducing potential using three-dimensional (3D) primary cultures of human hepatocytes (hepatocyte spheroids).

Evaluation of human hepatocytes cultured by three-dimensional spheroid systems for drug metabolism.

We investigated the utility of three-dimensional (3D) spheroid cultures of human hepatocytes in discovering drug metabolites. Metabolites of acetaminophen, diclofenac, lamotrigine, midazolam, propranolol and salbutamol were analyzed by liquid chromatography-tandem mass spectrometry (LC/MS/MS) to measure enzyme activities in this system cultured for 2 and 7 days. Sequential metabolic reactions by Phase I and then Phase II enzymes were found in diclofenac [CYP2C9 and UDP-glucuronyltransferases (UGTs)], midazolam (CYP3A4 and UGTs) and propranolol (CYP1A2/2D6 and UGTs).

Long-term survival and functional maintenance of hepatocytes by using a microfabricated cell array.

We developed a microfabricated cell array of hepatocyte spheroids that showed long-term viability and retained the properties of the parent hepatocytes. Fresh hepatocytes harvested from 8-week-old Wistar rats were cocultured with feeder cells to rapidly form hepatocyte spheroids; these cells retained the spheroidal formation for 42 days. We also evaluated the cellular functions of the hepatocytes such as albumin secretion and metabolic activity of cytochrome P450 (CYP).

Construction of Artificial Hepatic Lobule-Like Spheroids on a Three-Dimensional Culture Device.

One major purpose of cell culture is the reconstruction of physiological structures. Using bovine aortic epithelium cell line HH (JCRB0099) as feeder cells and rat primary hepatocytes, we constructed hepatic lobule-like spheroids on a cell array plate designed for three-dimensional (3D) culture. Microfabricated patterning of the cell array with poly(ethyleneglycol) brushes promotes the formation of spheroids at 100-μm diameter at 100-μm intervals.

Enhanced immunoresponse of antibody/mixed-PEG co-immobilized surface construction of high-performance immunomagnetic ELISA system.

Poly(ethylene glycol) possessing pentaethylenehexamine at one end (N6-PEG) was prepared via a reductive amination reaction of aldehyde-ended PEG with pentaethylenehexamine. Using N6-PEG, an antibody/PEG co-immobilized surface was constructed on magnetic particles via an active ester reaction method. After immobilization of the antibody on the active ester surface, N6-PEG was reacted on the magnetic beads.