State Key Laboratory of Bioelectronics, Southeast University, Nanjing 210096, China.
Biomaterials. 2010 May;31(13):3604-12. doi: 10.1016/j.biomaterials.2010.01.080. Epub 2010 Feb 9.
Primary hepatocytes cultured in three dimensional tissue constructs composed of multicellular aggregates maintain normal differentiated cellular function in vitro while cultured monolayers do not. Here, we report a technique to induce hepatocyte aggregate formation using type-I collagen-coated poly(L-lactic acid) (PLLA) discrete aligned nanofibers (disAFs) by providing limited cell-substrate adhesion strength and restricting cell migration to uniaxial movement. Kinetics of aggregate formation, morphology and biochemical activities of rat hepatocyte aggregates were tested over a 15 day culture period. Evidence was provided that physical cues from disAFs quickly induced the formation of aggregates. After 3 days in culture, 88.3% of free hepatocytes on disAFs were incorporated into aggregates with an average diameter of 61 +/- 18 microm. Hepatocyte aggregates formed on disAFs displayed excellent cell retention, cell activity and stable functional expression in terms of albumin secretion, urea synthesis and phase I and II (CYP1A and UGT) metabolic enzyme activity compared to monolayer culture of hepatocytes on tissue culture plastic (TCP) with type-I collagen as well as on meshes of type-I collagen-coated PLLA random nanofibers (meshRFs). These results suggest that disAFs may be a suitable method to maintain large-scale hepatic cultures with high activity for tissue engineering research and potential therapeutic applications, such as bioartificial liver devices.
在由多细胞聚集体组成的三维组织构建体中培养的原代肝细胞在体外保持正常的分化细胞功能，而单层培养则不能。在这里，我们报告了一种使用涂有 I 型胶原的聚 L-乳酸（PLLA）离散排列纳米纤维（disAF）诱导肝细胞聚集体形成的技术，通过提供有限的细胞-基底附着力和限制细胞迁移到单轴运动。在 15 天的培养期间测试了大鼠肝细胞聚集体的聚集动力学、形态和生化活性。有证据表明，disAFs 的物理线索可以快速诱导聚集的形成。培养 3 天后，disAFs 上的 88.3%游离肝细胞被整合到平均直径为 61 +/- 18 微米的聚集体中。与在组织培养塑料（TCP）上用 I 型胶原单层培养的肝细胞以及在涂有 I 型胶原的 PLLA 随机纳米纤维网（meshRFs）上的肝细胞相比，在 disAFs 上形成的肝细胞聚集体表现出优异的细胞保留、细胞活性和稳定的功能表达，表现在白蛋白分泌、尿素合成以及 I 期和 II 期（CYP1A 和 UGT）代谢酶活性方面。这些结果表明，disAFs 可能是一种适合维持具有高活性的大规模肝培养物的方法，用于组织工程研究和潜在的治疗应用，如生物人工肝装置。
