Animal Cell Technology Laboratory, ITQB-UNL/IBET, Oeiras, Portugal.
Tissue Eng Part C Methods. 2010 Dec;16(6):1223-32. doi: 10.1089/ten.TEC.2009.0784. Epub 2010 Apr 6.
The maintenance of differentiated hepatocyte phenotype in vitro depends on several factors-in particular, on extracellular matrix interactions, for example, with three-dimensional (3D) matrices. Alginate hydrogel provides the cells with a good extracellular matrix due to the formation of a massive capsule with semi-permeable properties that allows for diffusion of the medium components into the cells as well as efficient waste product elimination. Simultaneously, alginate protects the cells from shear stress caused by the hydrodynamics when cultured in stirred systems such as bioreactors. We have previously developed a hepatocyte aggregate 3D culture system in a bioreactor where improved hepatocyte functionality could be maintained over longer periods (21 days). In this work, ultra-high-viscosity alginate was used for hepatocyte aggregates entrapment. Hepatocyte biotransformation (phase I and II enzymes), CYP450 inducibility, and secretory capacity (albumin and urea production) were monitored. The analyses were performed in both spinner vessels and bioreactors to test the effect of the pO(2) control, unavailable in the spinners. Performance of alginate-encapsulated hepatocyte aggregates in culture was compared with nonencapsulated aggregate cultures in both bioreactor (controlled environment) and spinner vessels. For both culture systems, hepatocytes' metabolic and biotransformation capacities were maintained for up to 1 month, and encapsulated cells in bioreactors showed the best performance. In particular, albumin production rate increased 2- and 1.5-fold in encapsulated aggregates compared with nonencapsulated aggregates in bioreactor and spinner vessels, respectively. Urea production rate increased twofold in encapsulated cultures compared with nonencapsulated cells, in both bioreactor and spinner vessels. Similarly, in both the bioreactor and the spinner system, cell encapsulation resulted in a 1.5- and 2.8-fold improvement of hepatocyte 7-ethoxycoumarin and uridine diphosphate glucuronosyltransferases (UGT) activities, respectively. For all parameters, but for UGT activity, the bioreactor system resulted better than the spinner vessels; for UGT activity no difference was observed between the two. Furthermore, both encapsulated and nonencapsulated 3D culture systems were inducible by 3-methylcholanthrene and dexamethasone. The encapsulated systems consistently showed improved performance over the nonencapsulated cells, indicating that the protection conferred by the alginate matrix plays a relevant role in maintaining the hepatocyte functionalities in vitro.
体外维持分化的肝细胞表型取决于多种因素，特别是细胞外基质的相互作用，例如与三维（3D）基质的相互作用。藻酸盐水凝胶通过形成具有半透性的大量胶囊为细胞提供了良好的细胞外基质，允许介质成分扩散到细胞内，并有效地消除废物。同时，藻酸盐保护细胞免受搅拌系统（如生物反应器）中培养时由流体动力引起的剪切力。我们之前在生物反应器中开发了一种肝细胞聚集体 3D 培养系统，在该系统中可以维持较长时间（21 天）的改善的肝细胞功能。在这项工作中，使用超高粘度藻酸盐来包埋肝细胞聚集体。监测了肝细胞生物转化（I 期和 II 期酶）、CYP450 诱导能力和分泌能力（白蛋白和尿素的产生）。分析在搅拌器和生物反应器中进行，以测试在搅拌器中不可用的 pO2 控制的效果。将藻酸盐包封的肝细胞聚集体在培养中的性能与在生物反应器（受控环境）和搅拌器中的未包封聚集体培养进行比较。对于这两种培养系统，肝细胞的代谢和生物转化能力都可以维持长达 1 个月，并且生物反应器中的包封细胞表现出最佳性能。特别是，与生物反应器和搅拌器中的未包封聚集体相比，包封聚集体中的白蛋白产生速率分别增加了 2 倍和 1.5 倍。与未包封的细胞相比，包封培养物中的尿素产生速率增加了两倍，无论是在生物反应器还是在搅拌器中都是如此。同样，在生物反应器和搅拌器系统中，细胞包封分别使肝细胞 7-乙氧基香豆素和尿苷二磷酸葡萄糖醛酸基转移酶（UGT）活性提高了 1.5 倍和 2.8 倍。除 UGT 活性外，所有参数均显示生物反应器系统优于搅拌器系统；对于 UGT 活性，两者之间没有差异。此外，3-甲基胆蒽和地塞米松均可诱导包封和未包封的 3D 培养系统。包封系统始终表现出优于未包封细胞的性能，表明藻酸盐基质提供的保护在体外维持肝细胞功能方面发挥了重要作用。
