treatment: control treatment duration: 7 days hepatocyte vendor: TWJ hepatocyte lot: BioIVT group: Control Group
Treatment protocol
Cells were treated with control medium containing 1.0g/L glucose, 1.1 µM, and 10% bovine serum or steatosis inducing media containing 0.5 mM free fatty acid (FFA) (Palmitate/Oleate 2:1, Sigma), 10g/L glucose and 1.0g/L fructose (HGF), or a combination of FFA and HGF with media exchanges every other day. Cells were treated with steatosis inducers continuously until day 7 of treatment or switched back to control media on day 4 for 3 days, until day 7 of treatment (reversal samples).
Growth protocol
MPCC (HEPATOPAC) cultures were created using microfabrication processes to generate an organized co-culture containing cryopreserved primary human hepatocytes (PHH) from BioIVT (Baltimore, MD) and 3T3-J2 murine embryonic fibroblasts. Cryopreserved primary human hepatocytes from donor lot TWJ were thawed at 37°C, counted, and plated on collagen-patterned 96 or 24 well plates. The cells were allowed to adhere until the following day, when 3T3-J2 fibroblasts were introduced to the culture plates. The resulting culture contained multiple hepatocyte islands having a 500 μm diameter spaced 1200 μm apart, center-to-center; with 3T3-J2 murine fibroblasts filling the area surrounding the hepatocyte islands. MPCC cultures were stabilized in serum containing medium for 7 days. At maturity, approximately 3,100 hepatocytes and 15,000 fibroblasts were present/well in a 96 well plate and about 21,000 hepatocytes and 90,000 fibroblasts were present/well in a 24 well plate. Treatment was initiated on day 7 after initiation of culture manufacture.
Extracted molecule
total RNA
Extraction protocol
RNA was extracted from treated and control samples using the RNAeasy kit (Qiagen, Germany) as per manufacturer’s instructions. RNA was quantified spectrophotometrically, and all samples were normalized to the lowest concentration in the group. cDNA synthesis was performed using the RT2 First Strand Kit (Qiagen, Germany).
Label
SYBR green
Label protocol
PCR assays were performed using the RT2 Profiler PCR Array Human Fatty Liver (Qiagen, Germantown, MD) following the Manufacturer’s instructions. Reverse transcription was performed using the RT2 First Strand Kit (Qiagen, Germantown, MD). Quantitative real-time PCR were performed (Quantstudio5, Applied Biosystems, MA) with 40 cycles at 95 oC for 15 seconds and 60 oC for 60 seconds.
Hybridization protocol
n/a
Scan protocol
n/a
Description
Control
Data processing
Values for triplicate samples for treatment and 6 samples for control group were averaged for the analysis The normalization and all the data analysis were performed according to the manufacturers instructions using their web-based software package: https://geneglobe.qiagen.com/us/analyze For the normalization it uses the average of the arithmetic mean of five housekeeping genes: B2M, HPRT1 , RPLP0, GAPDH, ACTB. Target gene signals normalized to housekeeping genes; 2^-deltaCt, where deltaCt = (Ct_Target − Ct_HKG)] The web-based software package automatically performs all deltadeltaCt based fold-change calculations from the uploaded raw threshold cycle data. Data from triplicate samples (6 samples for control group) were grouped and the web based software determined fold change for each treatment group as compared to the control group Matrix normalized worksheet reports normalized signal (against housekeeping genes). Web based analysis software perfomed normalization automatically against housekeeping genes. Fold Change worksheet reports test/control (such as FFA/control) ratios.
