Collagen gel immobilization: A useful cell culture technique for long-term metabolic studies on human hepatocytes

Abstract

1. Primary cultures of human hepatocytes have already been employed in various applications for the study of xenobiotic metabolism. Most of these approaches were performed either on freshly isolated cells or on short-term primary cultures. Standard culture techniques do not maintain functional stability of P450 enzymes for > 1 week in vitro. 2. The aim of this study was to demonstrate the beneficial effect of an easy to apply, extracellular matrix configuration on the long-term performance of cultured human liver cells. Light microscopical examination of the cultures indicated that the cells remained viable over 1 month. As revealed by electron microscopy, hepatocytes exhibited bile canaliculi and desmosomes and were rich in mitochondria and endoplasmatic reticulum, indicating metabolic activity. 3. An early culture phase (3 days after isolation) could be described with decreasing DNA content of the cultures, peak values of alanine-amino-transferase (ALAT), and increasing albumin synthesis. After this adaptive period stable levels for DNA content and albumin synthesis were noted; ALAT returned to low values. 4. Functional activity was monitored by measurements of P450 1A1-dependent O-demethylation of p-nitroanisole to p-nitrophenol, which appeared to be constant over 3 weeks and weakly inducible by 1 mM phenobarbital. Another set-up examined conjugation of acetaminophen at subtoxic concentrations: acetaminophen was metabolized to its glucuronide and sulphate; 3-(glutathione-S-yl)-acetaminophen was not detected. Almost identical metabolism was found, comparing day 3 with 16 of culture. 5. We concluded that collagen gel immobilization not only provides mechanical support to cultured hepatocytes, but also supports long-term differentiated function of the cells for metabolic studies.

Regulation of the Major Detoxication Functions by Phenobarbital and 3‐Methylcholanthrene in Co‐Cultures of Rat Hepatocytes and Liver Epithelial Cells

“…Other models have been developed, such as the spheroid or sandwich methods, but they have been poorly described with regard to their potential to maintain drug metabolism capacities (Bader et al, 1994(Bader et al, , 1996Koebe et al, 1994;Niwa et al, 1996). In addition, no regulation studies have been reported as yet using these model systems.…”

Modeling the in Vitro Intrinsic Clearance of the Slowly Metabolized Compound Tolbutamide Determined in Sandwich-Cultured Rat Hepatocytes

“…Type I collagen was prepared from rat tail tendons as described by Koebe et al (1994) by the modified procedure of Elsdale and Bard (1972). This preparation yields type I collagen, mostly in its native, not cross-linked, triple-helical form (Beken et al, 1997a).…”

“…Other characteristics of this culture system are a cellular morphology comparable to the in vivo situation, including cellular polarity, physiological levels of protein secretion, development of an extensive bile canalicular network, and expression of multidrug resistance proteins. Furthermore, phase I enzyme activities remain better expressed for a few days compared with hepatocytes cultured without collagen (Koebe et al, 1994;Kern et al, 1997), whereas phase II glutathione S-transferase activity is stably expressed for over 14 days (Beken et al, 1997a;Lecluyse et al, 2000). Hence, the clearance of slowly metabolized compounds can easily be determined in this model by measuring the substrate depletion in the medium only.…”

Microbial P450 enzymes in biotechnology

“…It renders the enzymes stable for operation and storage (Tischer and Wedekind 1999) and allows simple product separation. So far, only few mammalian P450s have been immobilized (FernandezSalguero et al 1993;Koebe et al 1994a;Koebe et al 1994b), and reports on the immobilization of microbial P450 enzymes are rare. The first studies on the immobilization of a P450 enzyme from Saccharomyces cerevisiae were based on microsomal fractions (Azari and Wiseman 1982) or permeabilized cells (King et al 1988).…”