Comparative Study between the 3D‐Liver Spheroid Models Developed from HepG2 and Immortalized Hepatocyte‐Like Cells with Primary Hepatic Stellate Coculture for Drug Metabolism Analysis and Anticancer Drug Screening
Abstract:Liver spheroids may be the best alternative models for evaluating efficacy and toxicity of the new anticancer candidates and diagnostics for hepatocellular carcinoma (HCC). Here, novel 3D-liver spheroid models are constructed from human hepatoma cells (HepG2)/ immortalized human hepatocyte-like cells (imHCs) with primary hepatic stellate cells (HSCs) coculture using the ultralow attachment technique. Spheroid morphology, HSC distribution, metabolic activity, protein expressions, and drug penetration are evalua… Show more
“…Similar observations were made by other research groups. [24,37,38] They showed that the presence of stellate cells in the structure of the aggregate facilitates its compactness. 2D and 3D cocultures of hepatocytes and endothelial cells were also presented in the literature.…”
The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug‐induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three‐dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell–cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.
“…Similar observations were made by other research groups. [24,37,38] They showed that the presence of stellate cells in the structure of the aggregate facilitates its compactness. 2D and 3D cocultures of hepatocytes and endothelial cells were also presented in the literature.…”
The liver is one of the most important organs in the human body. It performs many important functions, including being responsible for the metabolism of most drugs, which is often associated with its drug‐induced damage. Currently, there are no ideal pharmacological models that would allow the evaluation of the effect of newly tested drugs on the liver in preclinical studies. Moreover, the influence of hepatic metabolism on the effectiveness of the tested drugs is rarely evaluated. Therefore, in this work we present an advanced model of the liver, which reflects most of the morphologically and metabolically important features of the liver in vivo, namely: three‐dimensionality, cellular composition, presence of extracellular matrix, distribution of individual cell types in the structure of the liver model, high urea and albumin synthesis efficiency, high cytochrome p450 activity. In addition, the work, based on the example of commonly used anticancer drugs, shows how important it is to take into account hepatic metabolism in the effective assessment of their impact on the target organ, in this case cancer. In our research, we have shown that the most similar to liver in vivo are 3D cellular aggregates composed of three important liver cells, namely hepatocytes (HepG2), hepatic stellate cells (HSCs), and hepatic sinusoidal endothelial cells (HSECs). Moreover, we showed that the cells in 3D aggregate structure need time (cell–cell interactions) to improve proper liver characteristic. The triculture model additionally showed the greatest ability to metabolize selected anticancer drugs.
“…The protein expression of albumin and mRNA expression of hepatocyte markers CK18 , HNF4 α, RBP4 , and CYP2E1 (with the exception of CYP3A4 ) in human 3D liver spheroids (HLS) are similar to those observed in human primary plated hepatocytes (2D) cultured for one day. In particular, the mRNA expression of CYP3A4 increased in a time-dependent manner, as HSCs could promote liver sinusoidal structure development and enhance the activity of CYP3A4 11 ( Figures 2 F and 2G). Figure 2 Characterization of normal human liver spheroids Immunofluorescent images of spheroids stained for (A) Hepatocyte nuclear factor 4α (HNF4α), Vimentin and DAPI; or (B) CD68, NCR1, PECAM, and DAPI (scale bar = 250 μm).…”
“…The reagent is designed to efficiently penetrate cells within a biological sample, including dense 3D structures like tumor spheroids [20,21]. Our choice was driven by the balance between methodological accessibility and the ability to compare our results with a broad range of existing literature [22]. Cells were treated with the stain solution at a concentration of 2.5 μM in the cell culture medium for 45 min.…”
The multifaceted nature of photodynamic therapy (PDT) requires a throughout evaluation of a multitude of parameters when devising preclinical protocols. In this study, we constructed MCF‐7 human breast tumor spheroid assays to infer PDT irradiation doses at four gradient levels for violet light at 408 nm and red light at 625 nm under normal and hypoxic oxygen conditions. The compacted three‐dimensional (3D) tumor models conferred PDT resistance as compared to monolayer cultures due to heterogenous distribution of photosensitizers along with the presence of internal hypoxic region. Cell viability results indicated that the violet light was more efficient to kill cells in the spheroids under normal oxygen conditions, while cells exposed to the hypoxic microenvironment exhibited minimal PDT‐induced death. The combination of 3D tumor spheroid assays and the multiparametric screening platform presented a solid framework for assessing PDT efficacy across a wide range of different physiological conditions and therapeutic regimes.
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