School of Chemical Engineering, Oklahoma State University, Stillwater, OK, 74078, USA.
J Appl Toxicol. 2019 Mar;39(3):461-472. doi: 10.1002/jat.3737. Epub 2018 Oct 11.
Recent advances in developing in vitro tissue models show that function of hepatocytes is altered in when cultured in 3D configuration and co-culturing with various non-parenchymal cells. However, tissue source for such non-parenchymal cells on viability and metabolic products of hepatocytes have not been explored. In this study, we evaluated the effect of 2D and 3D cultures either with HepaRG cells alone or in combination with liver sinusoidal endothelial cells (LSECs) and human umbilical vein ECs (HUVECs). For 3D cultures, we used chitosan-gelatin porous structures formed by freeze-drying. We cultured cells for 8 days before challenging with 1 mm acetaminophen (APAP) and assessed APAP, APAP-sulfate and APAP-glucuronide for 24 hours at 6-hour time intervals using high-performance liquid chromatography. We used multiple methods (phase contrast, confocal and scanning electron microscopy and histology via hematoxylin and eosin staining) to ensure cell distribution. We also measured total protein content and albumin secretion and viability. HUVEC 3D co-cultures showed the lowest HepaRG cell viability, while both 2D and 3D LSEC co-cultures had highest HepaRG cell viability. In addition, 3D cultures had significantly higher EC viability relative to 2D cultures. Further, HUVEC co-cultures showed reduced total protein content and albumin expression as early as day 4. However, urea production on a total protein content basis did not change. In addition, LSEC 3D co-cultures had the highest APAP conversion with reduced APAP-sulfate and APAP-glucuronide formation. CYP3A4 was higher in co-culture with HUVEC for 2D and 3D cultures. In conclusion, HepaRG cells with EC co-cultures demonstrated sensitivity to the EC line used.
最近在开发体外组织模型方面的进展表明，当肝细胞在 3D 结构中培养并与各种非实质细胞共培养时，其功能会发生改变。然而，用于这些非实质细胞的组织来源对肝细胞的活力和代谢产物尚未得到探索。在这项研究中，我们评估了 HepaRG 细胞单独培养或与肝窦内皮细胞（LSEC）和人脐静脉 EC（HUVEC）共培养的 2D 和 3D 培养的效果。对于 3D 培养，我们使用通过冷冻干燥形成的壳聚糖-明胶多孔结构。我们在挑战 1mm 对乙酰氨基酚（APAP）之前培养细胞 8 天，并在 6 小时时间间隔内使用高效液相色谱法评估 24 小时的 APAP、APAP-硫酸盐和 APAP-葡萄糖醛酸。我们使用多种方法（相差、共聚焦和扫描电子显微镜以及通过苏木精和伊红染色的组织学）来确保细胞分布。我们还测量了总蛋白含量、白蛋白分泌和活力。HUVEC 3D 共培养显示 HepaRG 细胞活力最低，而 2D 和 3D LSEC 共培养则具有最高的 HepaRG 细胞活力。此外，3D 培养的 EC 活力明显高于 2D 培养。此外，HUVEC 共培养早在第 4 天就显示出总蛋白含量降低和白蛋白表达减少。然而，基于总蛋白含量的尿素产生没有变化。此外，LSEC 3D 共培养具有最高的 APAP 转化率，减少了 APAP-硫酸盐和 APAP-葡萄糖醛酸的形成。CYP3A4 在与 HUVEC 的共培养中在 2D 和 3D 培养中更高。总之，与 EC 共培养的 HepaRG 细胞对所用的 EC 系表现出敏感性。
