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Pharmacology research & perspectives2016; 4(5); e00268; doi: 10.1002/prp2.268

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 (Cl) 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 × 10 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 Cl of three drugs used in equine medicine; omeprazole, flunixin, and phenylbutazone, via the substrate depletion method. Cryopreserved suspensions gave a comparable estimation of Cl 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.
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Publication Date: 2016-09-30 PubMed ID: 27713829PubMed Central: PMC5045944DOI: 10.1002/prp2.268Google Scholar: Lookup
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Summary

This research summary has been generated with artificial intelligence and may contain errors and omissions. Refer to the original study to confirm details provided. Submit correction.

This research explored the isolation, cryopreservation, and recovery of equine (horse) liver cells, or hepatocytes, bringing forth an effort to forge a ‘bank’ of these preserved cells for use in the estimation of drug metabolism rates in horses, in addition to producing horse-specific drug metabolites.

Isolation of Hepatocytes

  • The author’s study details the process of isolating primary equine hepatocytes which was done with a two-step collagenase perfusion method.
  • This method yielded an average of 2.47 ± 2.62 x 10 cells per gram of the perfused liver tissue with a viability rate of 84.1 ± 2.62%.

Cryopreservation and Recovery

  • The isolated hepatocytes were cryopreserved in a William’s medium E containing 10% fetal bovine serum with 10% DMSO, which is a cryoprotectant that prevents ice crystal formation in cells during the freezing process.
  • The viability of the cells was tested after their recovery from a 30% Percoll gradient, which allowed for the separation of viable and non-viable cells.
  • The viability of the recovered cells was 77 ± 11% and the estimated recovery rate was approximately 27%.

In Vitro Drug Metabolism Studies

  • The study also details the use of these cryopreserved equine hepatocytes in determining the in vitro clearance of three drugs commonly used in equine medicine. These were omeprazole, flunixin, and phenylbutazone.
  • The clearance was determined via the substrate depletion method which measures the decrease in concentration of the drug over time due to metabolism.
  • The cryopreserved cells were found to deliver comparable estimations of clearance as fresh cells did for these three drugs, and also produced the same metabolites.

Implications of the Study

  • The success of these procedures holds promise for the establishment of a ‘cell bank’ of cryopreserved equine hepatocytes.
  • This resource could help to estimate pharmacokinetic parameters such as the in vivo clearance of a drug in the horse’s liver.
  • It could also assist in producing horse-specific drug metabolites, which enhances the understanding of how different substances will be metabolized specifically by horses, increasing the efficiencies of drugs for equine use.

Cite This Article

APA
Shibany KA, Tötemeyer S, Pratt SL, Paine SW. (2016). Equine hepatocytes: isolation, cryopreservation, and applications to in vitro drug metabolism studies. Pharmacol Res Perspect, 4(5), e00268. https://doi.org/10.1002/prp2.268

Publication

Pharmacology research & perspectives
ISSN: 2052-1707
NlmUniqueID: 101626369
Country: United States
Language: English
Volume: 4
Issue: 5
Pages: e00268
PII: e00268

Researcher Affiliations

Shibany, Khaled A
  • School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom.
Tötemeyer, Sabine
  • School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom.
Pratt, Stefanie L
  • School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom.
Paine, Stuart W
  • School of Veterinary Medicine and Sciences University of Nottingham College Road Sutton Bonington Leicestershire LE12 5RD United Kingdom.

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Citations

This article has been cited 1 times.
  1. Nowak MR, Zdunek R, Pliński E, Świątek P, Strzelecka M, Malinka W, Plińska S. Recognition of Pharmacological Bi-Heterocyclic Compounds by Using Terahertz Time Domain Spectroscopy and Chemometrics. Sensors (Basel) 2019 Jul 30;19(15).
    doi: 10.3390/s19153349pubmed: 31366175google scholar: lookup
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