E.I. Goldie

Viability and function of cryopreserved rat hepatocyte monolayers as a function of cryopreservation media composition

Cytochrome P450 4A1 (CYP4A1) is involved in -hydroxylation of fatty acids and eicosanoids. The resulting metabolites have physiological activities such as regulation of blood pressure. However, little structural information of CYP4A1 is known. The C-helix region in CYP4A1, which is close to, and putatively makes contact with, the heme group in CYP4A1, is conserved among CYP4 proteins. However, the function of this region is still not understood. A model of CYP4A1 was constructed by homology modelling with the crystal structure of CYP102. Four residues including H141, R142, R143 and F149 in C-helix of CYP4A1 were chosen to study by site-directed mutagensis. H141 is located at the beginning of the C-helix, F149 is located at the end of C-helix, and these two residues appear to bind to conserved residues in the I-helix. R142 and R143 are in the middle of C-helix according to our computer model of CYP4A1, and may be involved in contacts with electron donors. We designed a series of mutants to test their function. For H141, three mutants including H141R, H141L and H141F were made. The H141R mutant retains the positive charge in the wild type but H141L and H141F mutants were changed to the neutral residues. For R142 and R143, the basic residue R was replaced by the neutral residue A. For F149, the aromatic residue F was changed to the alkyl I or to Y, which keeps the aromatic structure but carries the extra hydroxyl group. Therefore, seven mutants including H141R, H141F, H141L, R142A, R143A, F149I and F149Y were constructed. All mutants were expressed in E. coli. with an OmpA signal peptide. Expression of the R142A and F149I mutants yielded proteins of the expected size, but these proteins did not support a 450 nm peak in a reduced CO-difference spectrum, demonstrating an improperly folded enzyme. The other recombinant proteins were purified by Ni2+-chelate affinity chromatography, yielding homogenous protein with a correctly folded haem environment, as judged by the reduced CO-difference spectrum. The enzyme activity of these mutants for lauric acid metabolism is variable; preliminary data shows that the H141L and H141F have very poor enzyme activity, whereas the H141R retains enzyme activity. The results suggest that certain C-helix: I-helix contacts are not required for correct folding of the haem-environment, but are required for function of the P450 enzyme.To improve post-thaw attachment efficiency in culture exhibited by hepatocytes frozen in suspension, we have frozen rat hepatocytes as monolayers on collagen substrates and compared post-thaw viability and function of these cryopreserved monolayers with non-cryopreserved control cultures. To measure viability, carboxyfluorescein diacetate (CFDA) de-acetylation by intracellular esterases in viable cells was visualised under a confocal laser scanning microscope (CLSM). Function was assessed by the ability to metabolise kaempherol into two glucuronide metabolites by HPLC (Oliveira and Watson, 2000). Hepatocytes were prepared from male Sprague Dawley rats by collagenase perfusion (viability 80-90%). Petri dishes (60 mm) coated with 30 g/cm2 type I collagen isolated from rat tail tendons were seeded with 3×106 viable cells in 2 ml Chees medium (CM) containing 5% v/v foetal calf serum (FCS). Monolayer 24 h cultures of hepatocytes were frozen for 24 h at −70 °C and thawed according to Watts and Grant (1996), except that the cryopreservation (CP) medium was 10% DMSO in CM with 0-90% FCS present (see Fig. 1). Control cultures (24 h) were also treated with CP media, but not frozen. Media were changed in post-thaw and control cultures every 24 h thereafter. For viability assessment, 96 h control and post-thaw cultures were stained with 25 M CFDA for 20 min in the dark at 4 °C and imaged under CLSM. For functional assessment, 96 h cultures were incubated for 1 h with 100 M kaempherol in Krebs Hepes buffer and the metabolites analysed by HPLC.