In order to constitute a relevant hepatic in vitro system, cultured hepatocytes need to accurately reflect phenotypes and functionality seen in vivo. Importantly, expression of a selection of metabolic enzymes, drug and bile transporters and other important hepatic genes in spheroids pivoted around levels found in freshly isolated hepatocytes (Fig. 1a, Bell et al., SciRep, 2016). In contrast, transcripts of the same genes were rapidly lost in conventional 2D monolayer cultures from the same donors. Moreover, molecular phenotypes of 3D cultures closely resemble in vivo liver tissue on a proteomic level (n=5 donors), whereas proteomic signatures underwent wide-scale and rapid changes in 2D cultures (Fig. 1b,c). Strikingly, 3D samples clustered together with the corresponding in vivo liver pieces they were derived from highlighting the preservation of inter-individual differences in PHH spheroid culture and the ability to study inter-individual variability in hepatic function in an in vitro system (Fig. 1d). HepaPredict AB is the only company that offers this system, which provides a novel step into a more physiological hepatic system, for use in e.g. drug development.
When evaluating the functional implications of the proteome alterations using gene set enrichment analyses, it was found that mitochondrial function (p = 1*10−13), oxidative phosphorylation (p = 1*10−11) and the TCA cycle (p = 6*10−4) were significantly affected only in the early stages of 2D culture (24 h), while the effect on a multitude of other important pathways such as glycolysis (p = 1*10−6), gluconeogenesis (p = 6*10−5), ethanol degradation (p = 7*10−3) and protein ubiquitination (p = 7*10−3) persisted also after 7 d in 2D monolayer culture (Fig. 1e). Proteins involved in apoptosis signaling (p = 5*10−3) and the γ-glutamyl cycle (p = 0.04) were only found to be enriched after prolonged 2D culture (7 d). Strikingly, no pathway was found to be differentially expressed in the 3D spheroid culture (padj>0.05 for all pathways; Bell et al., 2017; Vorrink et al., 2017).
Figure 1: The 3D spheroid system closely resemble the in vivo liver hepatocytes. a , Expression levels of metabolic enzymes (CYP2C8, CYP2C9, CYP2D6, CYP3A4), drug (SLCO1B1) and bile transporters ( ABCB11), critical hepatic transcription factors (HNF4A ) and secretory products ( ALB) are significantly elevated between 14- and 1834-fold in the 3D spheroid system after 7d compared to 2D culture at the same time point. b , Heatmap visualizing whole proteome analysis of primary human liver samples (n=5) after 24 h and 7d in 2D monolayer culture compared to HepaPredict spheroids (7d HepaPredict). Only differentially expressed proteins (n=574 proteins, p<0.05, F-test) are shown. Note that in vivo liver samples (black) and spheroids (green) cluster closely together while the proteomes of samples cultured in 2D (24 h = blue; 7 d = red) are distinctly different. c , Principle component analysis separates proteomes from liver and PHH spheroids from 2D monolayer-cultured samples. d , In vivo phenotypes are preserved in 3D culture, with each of the 3D samples clustering with the respective liver piece from the same donor. e , Venn diagram showing differentially regulated pathways after 24h or 7d in 2D monolayer culture and after 7d in the 3D spheroid platform. Numbers in circles indicate numbers of differentially expressed proteins compared to liver with p<0.05. Extensive deregulation of a variety of important metabolic and signaling pathways is observed in 2D whereas the proteomes of 3D spheroid cultures closely resemble in vivo livers. Indicated p-values are after Benjamini-Hochberg multiple testing correction. Figure modified from Bell et al., Sci Rep , 2016.