Natarajan Vaishaali, Berglund Eric J, Chen Dorothy X, Kidambi Srivatsan
Department of Chemical and Biomolecular Engineering, University of Nebraska-Lincoln, NE, 68588, USA.
Nebraska Center for Materials and Nanoscience, University of Nebraska-Lincoln, NE, 68588, USA.
RSC Adv. 2015;5(99):80956-80966. doi: 10.1039/C5RA15208A. Epub 2015 Sep 14.
Liver fibrosis occurs as a consequence of chronic injuries from viral infections, metabolic disorders, and alcohol abuse. Fibrotic liver microenvironment (LME) is characterized by excessive deposition and aberrant turnover of extracellular matrix proteins, which leads to increased tissue stiffness. Liver stiffness acts as a vital cue in the regulation of hepatic responses in both healthy and diseased states; however, the effect of varying stiffness on liver cells is not well understood. There is a critical need to engineer models that mimic the liver stiffness corresponding to various stages of disease progression in order to elucidate the role of individual cellular responses. Here we employed polydimethyl siloxane (PDMS) based substrates with tunable mechanical properties to investigate the effect of substrate stiffness on the behavior of primary rat hepatocytes. To recreate physiologically relevant stiffness, we designed soft substrates (2 kPa) to represent the healthy liver and stiff substrates (55 kPa) to represent the diseased liver. Tissue culture plate surface (TCPS) served as the control substrate. We observed that hepatocytes cultured on soft substrates displayed a more differentiated and functional phenotype for a longer duration as compared to stiff substrates and TCPS. We demonstrated that hepatocytes on soft substrates exhibited higher urea and albumin synthesis. Cytochrome P450 (CYP) activity, another critical marker of hepatocytes, displayed a strong dependence on substrate stiffness, wherein hepatocytes on soft substrates retained 2.7 fold higher CYP activity on day 7 in culture, as compared to TCPS. We further observed that an increase in stiffness induced downregulation of key drug transporter genes (NTCP, UGT1A1, and GSTM-2). In addition, we observed that the epithelial cell phenotype was better maintained on soft substrates as indicated by higher expression of hepatocyte nuclear factor 4α, cytokeratin 18, and connexin 32. These results indicate that the substrate stiffness plays a significant role in modulating hepatocyte behavior. Our PDMS based liver model can be utilized to investigate the signaling pathways mediating the hepatocyte-LME communication to understand the progression of liver diseases.
肝纤维化是由病毒感染、代谢紊乱和酒精滥用等慢性损伤引起的。纤维化肝微环境（LME）的特征是细胞外基质蛋白过度沉积和异常周转，这导致组织硬度增加。肝硬度在健康和疾病状态下的肝脏反应调节中起着至关重要的作用；然而，不同硬度对肝细胞的影响尚不清楚。迫切需要构建能够模拟与疾病进展不同阶段相对应的肝脏硬度的模型，以阐明个体细胞反应的作用。在这里，我们使用具有可调机械性能的聚二甲基硅氧烷（PDMS）基底物来研究底物硬度对原代大鼠肝细胞行为的影响。为了重现生理相关硬度，我们设计了软底物（2 kPa）来代表健康肝脏，硬底物（55 kPa）来代表患病肝脏。组织培养板表面（TCPS）作为对照底物。我们观察到，与硬底物和TCPS相比，在软底物上培养的肝细胞在更长时间内表现出更分化和功能性的表型。我们证明，软底物上的肝细胞表现出更高的尿素和白蛋白合成。细胞色素P450（CYP）活性是肝细胞的另一个关键标志物，对底物硬度有很强的依赖性，其中软底物上的肝细胞在培养第7天时CYP活性比TCPS高2.7倍。我们进一步观察到，硬度增加会导致关键药物转运蛋白基因（NTCP、UGT1A1和GSTM - 2）的下调。此外，我们观察到，如肝细胞核因子4α、细胞角蛋白18和连接蛋白32的高表达所示，软底物上的上皮细胞表型得到更好的维持。这些结果表明，底物硬度在调节肝细胞行为中起着重要作用。我们基于PDMS的肝脏模型可用于研究介导肝细胞与LME通讯的信号通路，以了解肝脏疾病的进展。
