Toxicological Exploration of Collagenase and Benzonase on Rat Precision-Cut Liver Slices.

Precision-cut liver slices (PCLSs) are an ex vivo liver model that allows isolation of extracellular vesicles (EVs), which are nano-sized particles involved in intercellular communication. However, EV recovery from PCLSs is hindered by adhesion to the extracellular matrix and macromolecules such as extracellular DNA. Enzymatic treatments, such as collagenase (COL) and the endonuclease benzonase (BEN), can aid their release, but potential effects on the tissue are unclear. Here, the effect of these enzymes separately, as well as combined, was analyzed to determine their effects on morphology, protein content, and the proteome of the PCLS. The enzymes showed minimal effects on cell viability and protein content after two different incubation times, whereas morphological analysis confirmed collagen degradation by COL and COL(BEN), along with decreased ribosomal and secretory secretory protein levels. BEN decreased the levels of proteins related to protein synthesis and degradation. Increased levels of proteins related to detoxification and energy production were observed upon addition of COL, BEN, and COL(BEN). This study emphasizes the potential and risks for the use of collagenase and benzonase for EV isolation from the PCLS.