Gori Manuele, Simonelli Maria Chiara, Giannitelli Sara Maria, Businaro Luca, Trombetta Marcella, Rainer Alberto
Department of Engineering, Tissue Engineering Laboratory, Università Campus Bio-Medico di Roma, Rome, Italy.
National Research Council - Institute for Photonics and Nanotechnologies (CNR-IFN), Rome, Italy.
PLoS One. 2016 Jul 20;11(7):e0159729. doi: 10.1371/journal.pone.0159729. eCollection 2016.
Nonalcoholic fatty liver disease (NAFLD) is a chronic liver disease worldwide, ranging from simple steatosis to nonalcoholic steatohepatitis, which may progress to cirrhosis, eventually leading to hepatocellular carcinoma (HCC). HCC ranks as the third highest cause of cancer-related death globally, requiring an early diagnosis of NAFLD as a potential risk factor. However, the molecular mechanisms underlying NAFLD are still under investigation. So far, many in vitro studies on NAFLD have been hampered by the limitations of 2D culture systems, in which cells rapidly lose tissue-specific functions. The present liver-on-a-chip approach aims at filling the gap between conventional in vitro models, often scarcely predictive of in vivo conditions, and animal models, potentially biased by their xenogeneic nature.
HepG2 cells were cultured into a microfluidically perfused device under free fatty acid (FFA) supplementation, namely palmitic and oleic acid, for 24h and 48h. The device mimicked the endothelial-parenchymal interface of a liver sinusoid, allowing the diffusion of nutrients and removal of waste products similar to the hepatic microvasculature. Assessment of intracellular lipid accumulation, cell viability/cytotoxicity and oxidative stress due to the FFA overload, was performed by high-content analysis methodologies using fluorescence-based functional probes.
The chip enables gradual and lower intracellular lipid accumulation, higher hepatic cell viability and minimal oxidative stress in microfluidic dynamic vs. 2D static cultures, thus mimicking the chronic condition of steatosis observed in vivo more closely.
Overall, the liver-on-a-chip system provides a suitable culture microenvironment, representing a more reliable model compared to 2D cultures for investigating NAFLD pathogenesis. Hence, our system is amongst the first in vitro models of human NAFLD developed within a microfluidic device in a sinusoid-like fashion, endowing a more permissive tissue-like microenvironment for long-term culture of hepatic cells than conventional 2D static cultures.
非酒精性脂肪性肝病（NAFLD）是一种全球性的慢性肝病，范围从单纯性脂肪变性到非酒精性脂肪性肝炎，后者可能进展为肝硬化，最终导致肝细胞癌（HCC）。HCC是全球癌症相关死亡的第三大原因，需要将NAFLD作为潜在风险因素进行早期诊断。然而，NAFLD的分子机制仍在研究中。到目前为止，许多关于NAFLD的体外研究因二维培养系统的局限性而受阻，在这种系统中细胞会迅速丧失组织特异性功能。当前的芯片肝脏方法旨在填补传统体外模型（通常几乎无法预测体内情况）与动物模型（可能因其异种性质而存在偏差）之间的空白。
将HepG2细胞培养在微流控灌注装置中，在补充游离脂肪酸（FFA）（即棕榈酸和油酸）的条件下培养24小时和48小时。该装置模拟了肝血窦的内皮-实质界面，允许营养物质扩散并去除类似于肝微血管的代谢废物。通过使用基于荧光的功能探针的高内涵分析方法，评估由于FFA过载导致的细胞内脂质积累、细胞活力/细胞毒性和氧化应激。
与二维静态培养相比，该芯片在微流控动态培养中能够使细胞内脂质逐渐积累且积累量更低，肝细胞活力更高，氧化应激最小，从而更紧密地模拟了体内观察到的脂肪变性慢性状况。
总体而言，芯片肝脏系统提供了一个合适的培养微环境，与二维培养相比，是研究NAFLD发病机制更可靠的模型。因此，我们的系统是以类血窦方式在微流控装置中开发的首批人类NAFLD体外模型之一，与传统二维静态培养相比，为肝细胞长期培养提供了更宽松的类组织微环境。
