cell type: primary human hepatocytes assay type: in vitro study type: single dose toxicity strain: Hu8119+Hu1591+Hu1540 dose: DMSO 0.5 % dose duration: 3 days dose frequency: daily
After quick thawing in a water bath at 37°C, viability of the cells was checked by a Trypan blue (CAS no. 72-57-1, Sigma–Aldrich) exclusion test as instructed in the supplier’s protocol (Invitrogen, 2012). All viability scores after thawing were in agreement with those listed by the supplier. Before CsA treatment, cells were allowed to acclimatize for 48 hours. This is needed for the hepatocytes to restore an in vivo like cellular configuration and enzyme expression levels as optimally as possible. PHH were treated with 30 µM CsA for 5 days daily followed by a washout-period (terminating CsA-treatment) of 3 days.
Cryopreserved primary human hepatocytes (PHH) were purchased from Life Technologies. Cells were cultured in pre-coated 24-well plates (2,100,000 cells/sample) in a 2-layer collagen sandwich (A11428-02, Gibco), according to the supplier’s protocol (Invitrogen, 2012). The following culture media were used: Hepatocyte Thawing Medium (HTM) for thawing (CM7500, Gibco), Williams’ Medium E (1x, no phenol red) (A1217601, Gibco) + Cell Maintenance supplement B kit (CM4000, Gibco) for plating and incubation.
Cells were cultured in triplicate in 24 wells in a collagen sandwich layer. Following 5 days of repetitive daily exposureand thereupon a washout of 3 days, cells lysates were harvested for total RNA isolation after 3, 5 and 8 days. Therefore, medium was removed and PHH were harvested in Qiazol (Qiagen). Total RNA was isolated using a miRNeasy Mini Kit (Qiagen) according to the manufacturer’s protocol and followed by DNase I (Qiagen) treatment. Upon purification, RNA concentrations were measured by means of a NanoDrop® ND-1000 spectrophotometer (Thermo Scientific) at 260 and 280 nm. RNA quality and integrity were assessed by using automated gel electrophoresis on an Agilent 2100 Bioanalyzer system (Agilent Technologies). Only RNA samples which showed clear 18S and 28S peaks and with an RNA integrity number (RIN) higher than 6, were used. Samples were stored at -80ºC until RNA hybridization.
standard Agilent labeling kit
MicroRNA expression profiling was performed using Agilent Sureprint G3 Unrestricted Human miRNA V19 8 × 60 K microarrays. The hybridization was performed following standard protocols, after which the microarray slides were washed and scanned using a DNA microarray scanner (Agilent Technologies).
DNA microarray scanner
The scanned images were converted into TXT files using the Feature Extraction Software v10.7.3.1 from Agilent Technologies, which were imported in R 2.15.3 (http://www.r-project.org) for quality control with an in-house developed pipeline . Filtering and normalization was performed using AgiMicroRna , which applies a LIMMA approach for selection of DE-miRs. Total gene signals were log2-transformed and quantile-normalized. DE-miRs with an absolute FC of 1.5 or higher (i.e., average log2 ratio of < −0.58 or > 0.58) and an FDR-corrected p-value < 0.05 were considered statistically significant.