BioIVT, Baltimore, MD, USA.
BioIVT, Medford, MA, USA.
Sci Rep. 2023 Sep 22;13(1):15837. doi: 10.1038/s41598-023-42785-9.
Non-alcoholic fatty liver disease (NAFLD) is a highly prevalent, progressive disorder and growing public health concern. To address this issue considerable research has been undertaken in pursuit of new NAFLD therapeutics. Development of effective, high-throughput in vitro models is an important aspect of drug discovery. Here, a micropatterned hepatocyte co-culture (MPCC) was used to model liver steatosis. The MPCC model (HEPATOPAC) is comprised of hepatocytes and 3T3-J2 mouse stromal cells plated onto a patterned standard 96-well or 24-well plate, allowing the cultures to be handled and imaged in a standardized multi-well format. These studies employed high content imaging (HCI) analysis to assess lipid content in cultures. HCI analysis of lipid accumulation allows large numbers of samples to be imaged and analyzed in a relatively short period of time compared to manual acquisition and analysis methods. Treatment of MPCC with free fatty acids (FFA), high glucose and fructose (HGF), or a combination of both induces hepatic steatosis. MPCC treatment with ACC1/ACC2 inhibitors, as either a preventative or reversal agent, showed efficacy against FFA induced hepatic steatosis. Drug induced steatosis was also evaluated. Treatment with valproic acid showed steatosis induction in a lean background, which was significantly potentiated in a fatty liver background. Additionally, these media treatments changed expression of fatty liver related genes. Treatment of MPCC with FFA, HGF, or a combination reversibly altered expression of genes involved in fatty acid metabolism, insulin signaling, and lipid transport. Together, these data demonstrate that MPCC is an easy to use, long-term functional in vitro model of NAFLD having utility for compound screening, drug toxicity evaluation, and assessment of gene regulation.
非酒精性脂肪性肝病（NAFLD）是一种高度流行且不断进展的疾病，也是日益受到关注的公众健康问题。为了解决这一问题，人们进行了大量研究，以期开发出新的 NAFLD 治疗方法。开发有效的高通量体外模型是药物发现的一个重要方面。在这里，我们使用微图案化肝细胞共培养物（MPCC）来模拟肝脂肪变性。MPCC 模型（HEPATOPAC）由肝细胞和 3T3-J2 小鼠基质细胞铺在图案化的标准 96 孔或 24 孔板上组成，使培养物能够以标准化的多孔板格式进行处理和成像。这些研究采用高内涵成像（HCI）分析来评估培养物中的脂质含量。与手动获取和分析方法相比，HCI 分析脂质积累可以在相对较短的时间内对大量样本进行成像和分析。用游离脂肪酸（FFA）、高葡萄糖和果糖（HGF）或两者的组合处理 MPCC 会诱导肝脂肪变性。用 ACC1/ACC2 抑制剂（作为预防剂或逆转剂）处理 MPCC 可有效抵抗 FFA 诱导的肝脂肪变性。还评估了药物诱导的脂肪变性。用丙戊酸钠处理显示在瘦肉背景下诱导脂肪变性，在脂肪肝背景下显著增强。此外，这些培养基处理还改变了与脂肪肝相关的基因表达。用 FFA、HGF 或两者的组合处理 MPCC 可可逆地改变参与脂肪酸代谢、胰岛素信号传导和脂质转运的基因的表达。总之，这些数据表明 MPCC 是一种易于使用的、长期的 NAFLD 功能性体外模型，可用于化合物筛选、药物毒性评估和基因调控评估。
