Research paperCryopreserved human hepatocytes in suspension are a convenient high throughput tool for the prediction of metabolic clearance
Introduction
The pharmaceutical industry is confronted with an increasing number of compounds, which are being synthesised weekly together with the need to test them at high speed and with a high degree of accuracy. In vitro clearance data have, traditionally, been generated from human and rat liver microsomal incubations, but such subcellular fractions do not represent the whole metabolic process that occurs in the liver, prompting researchers to move towards hepatocyte incubations.
Nowadays, human hepatocytes are recognised as the most reliable model to predict hepatic metabolic clearance (CLH) in humans [2], [3]. Until recently, hepatocytes were routinely used in primary culture. However, there are scientific limitations inherent to this configuration, such as decrease of phase I and phase II activities during culture [1], [4], [5], at least in part responsible for the observed tendency to under-predict in vivo hepatic clearance [6]. With the aim of improving clearance prediction, in vitro methods using rat [1], [7] and human [8], [9], [10] (Blanchard et al., submitted4) hepatocytes in suspension in 24- and 48-well plates were developed. Cytochrome P450 (CYP)-dependent activities (mainly CYP 3A1) were shown to be more stable during the first 6 h of incubation of rat hepatocytes in suspension than in primary cultures [1]. Moreover, this system presents the advantage of reducing the time required for preparation (cell plating and cell scraping are not required) and for incubation (performed only up to 4–6 h in suspension versus 20 h in primary culture).
A number of factors, such as inter-individual variability and the erratic and unpredictable availability of human liver samples, complicate the prediction of in vivo rates of metabolism from in vitro data in humans. At present, cryopreserved human hepatocytes are easily available and have been reported to retain, quantitatively, most of the phase I and phase II metabolic activities of fresh liver [11], [12]. We have shown recently [10] that clearances obtained from suspensions of fresh, cold-preserved (i.e., preserved for 20 h at 4 °C) or cryopreserved human hepatocytes in 24-well plates were similar, making these cells an easy-to-use system for CL determination.
Increasing throughput via automation, decreased cell consumption and the possibility to evaluate variability between humans requires the use of human hepatocytes in suspension in a miniaturised system. The aim of the present study, therefore, was to assess the prediction of hepatic clearance using: (1) freshly isolated rat and human hepatocytes in suspension in 96-well plates, (2) cold- and/or cryopreserved hepatocytes in this miniaturised system and (3) cryopreserved hepatocytes from various donors and from various sources (i.e., in house isolated or commercially available). For this, six reference compounds covering a broad range of clearance, and which were metabolised by a variety of CYP450 and phase II enzymes, namely theophylline, diclofenac, mibefradil, bosentan, bufuralol and midazolam were used.
Section snippets
Materials
Twenty-four-well plates (Falcon Cat. No 351147), 96-well plates (NUNC Microwell 442587), mixer (IKA HS/KS 260 control), Hepes buffer, William’s E w/o l-glutamine w/o phenol red (Gibco 041-94198M) and Dulbecco’s modified Eagle’s medium (DMEM) (Gibco 31053-028) were from Life Technologies AG, Basel, Switzerland, or Sigma W1878 (Division of Fluka Chemie AG, Buchs, Switzerland). Leibovitz L-15 medium, Percoll solution and insulin (Sigma I1882) were all purchased at Sigma. Penicillin/streptomycin
In vitro CLint and predicted in vivo CLH obtained using conventional primary cultures (CPC) and suspensions (FSH) of freshly isolated rat hepatocytes
A higher interassay variability of intrinsic clearance was observed with suspensions as compared to primary cultures (except for theophylline, a very low CL compound) (Table 2). However, no major discrepancies were obtained between primary cultures and suspensions with respect to both the in vitro hepatic clearances and the resulting in vivo hepatic clearances (Fig. 1). Thus, both hepatocyte models predict, well, the in vivo hepatic clearances of diclofenac, bosentan, bufuralol and
Discussion
Because of the increasing number of compounds synthesised weekly, the pharmaceutical industry needs to develop systems to speed up screening and ranking, in order to test new chemical entities with a higher throughput and a high degree of accuracy. Traditionally, human hepatocytes were used in primary cultures but were limited by both scientific issues such as their tendency to under-predict CLH and technical issues such as the erratic and unpredictable availability of human liver samples for
Acknowledgements
The authors thank F. Avenel, D. Doppler, C. Flament, S. Masur, I. Walter and Y. Siegrist for their analytical assistance.
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2016, Toxicology LettersCitation Excerpt :This has been partly attributed to the regeneration of co-factors in culture, suggesting a shift of phase I/phase II ratio towards phase II in cultured hepatocyes. Human hepatocytes in suspension, either freshly isolated or after cryopreservation, present a transcriptomic profile similar to liver in vivo (Richert et al., 2006), and their use in suspension has become a widely accepted model for prediction of in vivo metabolism (Jouin et al., 2006), drug–drug interactions through cytochrome P450 inhibition (Mao et al., 2012; Desbans et al., 2014) and have been used for assessment of drug and metabolite toxicity (Elaut et al., 2006). Although a major draw-back of the use of hepatocytes in suspension is their short life span, i.e. up to several hours, limiting the contact time of TCs with cells, pools of cryopreserved primary human hepatocytes used in suspension have been recently described as useful for the screening of hepatotoxicants (Mennecozzi et al., 2015).
Revival, characterization, and hepatitis B virus infection of cryopreserved human fetal hepatocytes
2014, Journal of Virological MethodsCitation Excerpt :Cell cryopreservation is known as a promising solution to these limitations. A large number of studies of methods of cryopreservation have been reported, aimed at providing a cell bank for fundamental studies, such as metabolism and cytotoxicity of xenobiotics, and clinical applications such as cell transplantation for patients suffering from acute or chronic hepatic failure (Rijntjes et al., 1986; Chesne et al., 1993; Coundouris et al., 1993; Li et al., 1999; Alexandre et al., 2002; Garcia et al., 2003; Terry et al., 2005; Jouin et al., 2006; Hang et al., 2009). According to preliminary experiments, cryopreserved primary human adult hepatocytes could not be well revived and cultured in our system (unpublished data).
A comprehensive evaluation of metabolic activity and intrinsic clearance in suspensions and monolayer cultures of cryopreserved primary human hepatocytes
2012, Journal of Pharmaceutical SciencesCitation Excerpt :Advantages of cryopreserved hepatocytes include lessened dependence on the availability of fresh tissues, convenience because of the ability to plan studies in accordance with timelines, ability to tailor studies by choice of specific lot characteristics, and the use of one particular batch for several assessments.13 Cryopreserved human hepatocytes are used routinely in 96‐well formats, demonstrating their utility in higher throughput metabolic screening.14 Although cryopreserved hepatocytes are now accepted for all metabolism‐based evaluations, direct comparisons of metabolic function using freshly isolated and cryopreserved human hepatocytes prepared from the same donor are limited.
A practical and direct comparison of intrinsic metabolic clearance of several non-CYP enzyme substrates in freshly isolated and cryopreserved hepatocytes
2012, Drug Metabolism and Pharmacokinetics
- 1
Present address: ADDEX Pharmaceuticals SA, Chemin des Aulx, 12-CH-1228 Plan-les-Ouates, Switzerland.
- 2
These two authors contributed equally to the present work.
- 3
Present address: KaLy Cell, Faculté de Pharmacie, 25030 Besançon, France.