The use of primary hepatocytes is now well established for both studies of drug metabolism and enzyme induction. Cryopreservation of primary hepatocytes decreases the need for fresh liver tissue. This is especially important for research with human hepatocytes because availability of human liver tissue is limited. In this review, we summarize our research on optimization and validation of cryopreservation techniques. The critical elements for successful cryopreservation of hepatocytes are (1) the freezing protocol, (2) the concentration of the cryoprotectant [10% dimethyl-sulfoxide (DMSO)], (3) slow addition and removal of DMSO, (4) carbogen equilibration during isolation of hepatocytes and before cryopreservation, and (5) removal of unvital hepatocytes by Percoll centrifugation after thawing. Hepatocytes of human, monkey, dog, rat, and mouse isolated and cryopreserved by our standard procedure have a viability > or = 80%. Metabolic capacity of cryopreserved hepatocytes determined by testosterone hydroxylation, 7-ethoxyresorufin-O-de-ethylase (EROD), 7-ethoxycoumarin-O-deethylase (ECOD), glutathione S-transferase, UDP-glucuronosyl transferase, sulfotransferase, and epoxide hydrolase activities is > or = 60% of freshly isolated cells. Cryopreserved hepatocytes in suspension were successfully applied in short-term metabolism studies and as a metabolizing system in mutagenicity investigations. For instance, the complex pattern of benzo[a]pyrene metabolites including phase II metabolites formed by freshly isolated and cryopreserved hepatocytes was almost identical. For the study of enzyme induction, a longer time period and therefore cryopreserved hepatocyte cultures are required. We present a technique with cryopreserved hepatocytes that allows the induction of testosterone metabolism with similar induction factors as for fresh cultures. However, enzyme activities of induced hepatocytes and solvent controls were smaller in the cryopreserved cells. In conclusion, cryopreserved hepatocytes held in suspension can be recommended for short-term metabolism or toxicity studies. Systems with cryopreserved hepatocyte cultures that could be applied for studies of enzyme induction are already in a state allowing practical application, but may be further optimized.
A knowledge of the fate of a drug, its disposition (absorption, distribution, metabolism, and excretion, known by the acronym ADME) and pharmacokinetics (the mathematical description of the rates of these processes and of concentration-time relationships), plays a central role throughout pharmaceutical research and development. These studies aid in the discovery and selection of new chemical entities, support safety assessment, and are critical in defining conditions for safe and effective use in patients. ADME studies provide the only basis for critical judgments from situations where the behavior of the drug is understood to those where it is unknown: this is most important in bridging from animal studies to the human situation. This presentation is intended to provide an introductory overview of the life cycle of a drug in the animal body and indicates the significance of such information for a full understanding of mechanisms of action and toxicity.
1. Dog hepatocytes were cryopreserved at 6 x 10(6) viable cells/ml in a suspension buffer containing 10% DMSO and were stored in liquid nitrogen. 2. The exclusion of trypan blue dye was 96 +/- 2 and 85 +/- 9% in fresh and cryopreserved (CP) hepatocytes, respectively. Albumin synthesis was unaffected by freezing. 3. Ethoxycoumarin and ethoxyresorufin O-deethylase activities were equivalent in fresh and CP hepatocytes. 4. The profile of testosterone metabolism was unaffected by freezing. Total hydroxylase activities were 815 +/- 33 pmol/min/10(6) cells in freshly isolated whole hepatocytes and 463 +/- 24 pmol/min/10(6) CP whole hepatocytes, but they were equivalent in fresh and CP hepatocyte homogenates supplemented with 250 microM NADPH. 5. Phase 2 enzymes were functional in freshly thawed CP hepatocytes but they required exogenous addition of cofactors (20 microM UDPGA and 1.7 microM PAPS). 6. When placed in suspension for longer times, fresh and CP cell viabilities were 88 +/- 6 and 64 +/- 2% after 4 h. ECOD and EROD activities were equivalent in fresh and CP hepatocyte suspensions, over 4 h. Testosterone hydroxylase activities were well maintained in fresh cell suspensions but they declined to 63 +/- 6% of the initial activity after 4 h in CP hepatocytes. 7. These results indicate that CP dog hepatocytes are a suitable in vitro system for xenobiotic metabolism since enzyme functions in CP hepatocytes were stabilized. Cofactors in freshly thawed CP hepatocytes should be measured and controlled for optimal use.
This small cohort shows that some cats with OM can be successfully managed medically. Surgery is invasive and may not necessarily be required if appropriate medical management is undertaken. This is the first study of OM treatment in cats and provides the basis for further studies, which should aim to establish specific infectious causes of OM and how they can potentially be managed with medical therapies.
Objectives Otitis externa is seen clinically in cats, although studies investigating this condition within the UK are lacking. The objective of this study was to investigate the prevalence of Otodectes cynotis mites and microbial infection in the ear canals of cats in various rescue centres and a referral hospital. Methods Otoscopy was performed in 332 cats. Otoscopic findings were noted, including the gross visualisation of Otodectes species. A sample of cerumen was collected for cytological evaluation and a cerumen smear for detection of Otodectes mites if there was a large amount of aural exudate present. Results O cynotis infestation was noted in 3/341 cats (0.9%, 95% confidence interval [CI] 0.3–2.6). A total of 129/341 (37.8%; 95% CI 32.7–43.0) cats were found to have Malassezia species within one or both ears. Bacteria were found unilaterally in 9/341 (2.6%; 95% CI 1.4–4.9) cats. Analysis of the cytological findings showed an increased likelihood for Malassezia species to be present as age increased (n = 293; Pearson r = 0.204, P <0.001). There was also an increased likelihood of finding Malassezia species in both ears if found within one ear (n = 327; r = 0.499, P <0.001). There was a positive correlation between the number of Malassezia organisms and the quantity of aural exudate (n = 338; r = 0.778, P <0.001). Cats in which Otodectes species infestation were noted (n = 3) had moderate or large quantities of cerumen. Conclusions and relevance This study shows that there was a low prevalence of O cynotis in this cohort of cats. In normal cats it was not unusual to find Malassezia microorganisms upon aural cytology, bacteria were noted far less frequently and in two cats this was associated with underlying anatomical pathology.
scite is a Brooklyn-based organization that helps researchers better discover and understand research articles through Smart Citations–citations that display the context of the citation and describe whether the article provides supporting or contrasting evidence. scite is used by students and researchers from around the world and is funded in part by the National Science Foundation and the National Institute on Drug Abuse of the National Institutes of Health.
hi@scite.ai
10624 S. Eastern Ave., Ste. A-614
Henderson, NV 89052, USA
Copyright © 2024 scite LLC. All rights reserved.
Made with 💙 for researchers
Part of the Research Solutions Family.