## 4 Discussion
The use of primary liver cell cultures is quite complex since they lose most of their characteristic functions when expanded in vitro. The HepG2 cell line maintains many differentiated hepatic functions and is a widely used in vitro model in the study of hepatocyte biology [25]. MSCs, on the other hand, are cells involved in the maintenance of tissue homeostasis, control of inflammatory processes, and regeneration of damaged tissues [40]. MSCs are distributed in all tissues of mesenchymal origin and playtheir trophic and control role towards other cells in physiological conditions, as well as in various diseases [41]. MSCs have demonstrated a multi-differentiative capacity that has also been observed towards the hepatocyte lineage, thus suggesting their application in treating liver diseases using tissue engineering and regenerative medicine approaches [10]. Biological preparations derived from liver tissue can provide adequate biochemical cues to maintain hepatic functions [10; 11; 42]. In particular, [PERSON] et al. [11] demonstrated that porcine-liver-derived powder maintained primary hepatocyte function in vitro at levels comparable to Matrigel, considered the most helpful substitute to maintain hepatic cells biological features in vitro. Concerning this, it has recently been proposed that liver extracts can modulate the hepatocyte differentiation of At-MSCs, suggesting an interplay between hepatic tissue and MSCs [43]. The colorimetric MTT assay was used to assess the cellular metabolic activity in response to LLP treatment. The results indicate that, although the effect is more potent on At-MSCs, the LLP exerts a statistically significant trophic effect on both cell lines. Since MTT assay is based on the measurement of mitochondrial respiration [44], it does not provide direct information about the replication activity of the cultured cells (i.e., it cannot be considered a direct test of cell proliferation); it rather allowed evaluation of the lack of cytotoxicity of the LLP on these cell lines at the different concentration tested. Although a precise dose-effect curve could not be determined, the effect reached saturation at treatment with 200-500 \(\upmu\)g of LLP for both cell lines, while the MTT signal was slightly lower at 1000 \(\upmu\)g. Interestingly, a dose-response relationship was observed for HepG2 cells expanded in serum-free medium supplemented with LLP. In this case, exposure to 200, 500, and 1000 \(\upmu\)g of the powder induced a cellular metabolic activity comparable to what was observed for culture medium supplemented with 10% FBS. The bromo-deoxyuridine (BrdU) incorporation assays confirmed a direct effect of the LLP on the in vitro replication of HepG2 and At-MSCs, although a difference was observed between the two cell types. Regarding the HepG2 cell line, a stimulatory effect was observed only for the treatment with 200 \(\upmu\)g, while no effect was present with the 500 \(\upmu\)g of LLP treatment. A more evident effect was observed on At-MSCs, where both 200 and 500 \(\upmu\)g LLP supplementation induced a significant stimulus on DNA synthesis. Since no difference was observed between the dosages, it can be hypothesized that a plateau effect had already been reached with the 200 \(\upmu\)g treatment. To confirm the effects of LLP on cell proliferation, direct cell counts were performed after treating the two cell lines with 100, 200, and 500 \(\upmu\)g. The results underlined a different response of cells to LLP. While a limited, not statistically significant, increase in cell number was observed for HepG2 treated with 100 and 200 \(\upmu\)g, a statistically significant increase was observed in At-MSCs for the three different concentrations tested (\(p<0.001\)). Furthermore, the cell-doubling time was significantly lower for At-MSCs treated with LLP, confirming its stimulatory effect on cell proliferation. Direct cell count excluded a cytotoxic effect on both cell lines.