Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies

Abstract

Despite reports of the successful isolation of primary equine hepatocytes, there are no published data regarding the successful cryopreservation of these isolated cells. In this study, a detailed description of the procedures for isolation, cryopreservation, and recovery of equine hepatocytes are presented. Furthermore, the intrinsic clearance (Clint) and production of metabolites for three drugs were compared between freshly isolated and recovered cryopreserved hepatocytes. Primary equine hepatocytes were isolated using a two‐step collagenase perfusion method, with an average cell yield of 2.47 ± 2.62 × 106 cells/g of perfused liver tissue and viability of 84.1 ± 2.62%. These cells were cryopreserved with William's medium E containing 10% fetal bovine serum with 10% DMSO. The viability of recovered cells, after a 30% Percoll gradient, was 77 ± 11% and estimated recovery rate was approximately 27%. These purified cells were used to determine the in vitro Clint of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Clint compared to fresh cells for these three drugs as well as producing the same metabolites. This work paves the way for establishing a bank of cryopreserved equine hepatocytes that can be used for estimating pharmacokinetic parameters such as the hepatic metabolic in vivo clearance of a drug as well as producing horse‐specific drug metabolites.

Recognition of Pharmacological Bi-Heterocyclic Compounds by Using Terahertz Time Domain Spectroscopy and Chemometrics

“…They belong to a group of bi-heterocycles with a common core based on isothiazolpyridine derivatives, more precisely 4,6-dimethyl-2,3-dihydroisothiazolo[5,4-b]pyridin-3-on (Figure 2, last item, in blue). These are compounds inhibiting cyclooxygenases COX-1 and COX-2 [58,59] and are still in the ‘in vitro’ testing phase. Drugs that have similar properties but are registered and are commonly found in the pharmaceutical market are referred to as non-steroidal anti-inflammatory drugs (NSAIDs).…”

Innovation for hepatotoxicity in vitro research models: A review

“…Various media supplements such as amino acids, hormones, growth factors, and other co-factors are important in primary culture. Dulbecco's modified Eagle's medium (Cho et al, 2015;Nemoto, Sakurai, Tazawa, & Ishikawa, 1989;Nikoozad, Ghorbanian, & Rezaei, 2014;Wu et al, 2015;Zhang et al, 2014), Williams' E (Shibany, Totemeyer, Pratt, & Paine, 2016;Shri, Agrawal, Rani, Singh, & Onteru, 2017;Tomizawa et al, 2016b), Waymouth's MB (Rodriguez-Enriquez, Kai, Maldonado, Currin, & Lemasters, 2009), Ham's F12 (Rattanasinganchan et al, 2006;Sripa et al, 2005), RPMI 1640 (Horai et al, 2014;Khosravi, Shokri, & Eshghi, 2017;Mehdizadeh et al, 2017) and Leibovitz's 15 (L-15) (Anene, Rosenberg, Kleiner, Cornish, & Halushka, 2016;Mitaka, Sattler, & Pitot, 1991) are all available media formulations. Highly enriched media, which contain amino acid concentrations that are 5-10 times higher than most standard media, are superior for the maintenance of cell survival, preserving cellular protein levels and liver-specific functions (Gebhardt et al, 2003;Swift, Pfeifer, & Brouwer, 2010).…”

The effects of aging on hepatic microsomal scaling factor and hepatocellularity number in the horse

“…Homogenization of hepatocyte suspensions. Fresh hepatocyte suspensions of known cell concentration were cryopreserved as previously described (Shibany et al 2016). Cryopreserved hepatocytes were thawed and the total number of cells was recounted.…”