MASH human liver - secreted proteome

An approximate 1 cm3 wedge liver biopsy was collected from the left lobe of the liver during surgery. The liver was cut into two portions. One portion was placed in formalin and transported to TissuPathTM (Mount Waverley, Victoria), paraffin embedded and processed for histological analysis. Samples were graded according to the Clinical Research Network (CRN) NAFLD activity score (NAS) [36] and Kleiner classification of liver fibrosis [4] by TissuPath or Alfred Pathology. The main outcome was the diagnosis of NASH CRN where steatosis, inflammation, and ballooning scores are ≥1. The other portion of liver was placed in oxygenated Medium 199 media (M199; Gibco, USA) with 10% fetal bovine serum (FBS; Gibco, USA) and 1% penicillin-streptomycin (P/S; Gibco, USA), embedded in 3% SeaPlaque agarose (Lonza BioScience, USA) using a Tissue Embedding Unit (Alabama Research and Development, USA). Embedded livers were then sliced on the Alabama R&D Tissue Slicer (Alabama Research and Development, USA) in 500 mL of oxygenated phenol red-free Dulbecco's Modified Eagle Medium (DMEM; Gibco, USA) with 1% P/S at 300 µm thickness, as previously described [24, 37, 38]. The liver slices were washed in Phosphate Buffered Saline (PBS; Gibco USA) and cultured in oxygenated M199 medium containing 1% P/S for 1 h. Subsequently, the liver slices were washed with PBS and cultured in 1 mL EX-CELL 325 protein free medium (Sigma-Aldrich, Australia) for 16 h at 37oC. The following day, the liver slices were weighed and snap-frozen in liquid nitrogen, while the incubation medium was collected and centrifuged at 300 x g at 4 ºC for 10 min. The supernatant was snap-frozen and stored at -80 ºC for subsequent proteomics analysis.