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Optimized Methods to Explore the Mechanistic and Biomarker Potential of Hepatocyte-Derived Exosomes in Drug-Induced Liver Injury.
- Source :
-
Toxicological sciences : an official journal of the Society of Toxicology [Toxicol Sci] 2018 May 01; Vol. 163 (1), pp. 92-100. - Publication Year :
- 2018
-
Abstract
- Recent evidence supports that alterations in hepatocyte-derived exosomes (HDE) may play a role in the pathogenesis of drug-induced liver injury (DILI). HDE-based biomarkers also hold promise to improve the sensitivity of existing in vitro assays for predicting DILI liability. Primary human hepatocytes (PHH) provide a physiologically relevant in vitro model to explore the mechanistic and biomarker potential of HDE in DILI. However, optimal methods to study exosomes in this culture system have not been defined. Here we use HepG2 and HepaRG cells along with PHH to optimize methods for in vitro HDE research. We compared the quantity and purity of HDE enriched from HepG2 cell culture medium by 3 widely used methods: ultracentrifugation (UC), OptiPrep density gradient ultracentrifugation (ODG), and ExoQuick (EQ)-a commercially available exosome precipitation reagent. Although EQ resulted in the highest number of particles, UC resulted in more exosomes as indicated by the relative abundance of exosomal CD63 to cellular prohibitin-1 as well as the comparative absence of contaminating extravesicular material. To determine culture conditions that best supported exosome release, we also assessed the effect of Matrigel matrix overlay at concentrations ranging from 0 to 0.25 mg/ml in HepaRG cells and compared exosome release from fresh and cryopreserved PHH from same donor. Sandwich culture did not impair exosome release, and freshly prepared PHH yielded a higher number of HDE overall. Taken together, our data support the use of UC-based enrichment from fresh preparations of sandwich-cultured PHH for future studies of HDE in DILI.
- Subjects :
- Adult
Collagen chemistry
Cryopreservation
Culture Media chemistry
Drug Combinations
Exosomes metabolism
Female
Hep G2 Cells
Humans
Laminin chemistry
Male
Middle Aged
Primary Cell Culture
Prohibitins
Proteoglycans chemistry
Repressor Proteins analysis
Tetraspanin 30 analysis
Biomarkers analysis
Chemical and Drug Induced Liver Injury
Coculture Techniques methods
Exosomes ultrastructure
Hepatocytes cytology
Ultracentrifugation methods
Subjects
Details
- Language :
- English
- ISSN :
- 1096-0929
- Volume :
- 163
- Issue :
- 1
- Database :
- MEDLINE
- Journal :
- Toxicological sciences : an official journal of the Society of Toxicology
- Publication Type :
- Academic Journal
- Accession number :
- 29385596
- Full Text :
- https://doi.org/10.1093/toxsci/kfy015