Department of Hepatobiliary and Transplant Surgery, University Medical Center Hamburg-Eppendorf, Martinistrasse 52, 20246 Hamburg, Germany.
Biotechnol Bioeng. 2011 Jan;108(1):141-50. doi: 10.1002/bit.22924.
Primary rat hepatocytes are a widely used experimental model to estimate drug metabolism and toxicity. In currently used two-dimensional (2D) cell culture systems, typical problems like morphological changes and the loss of liver cell-specific functions occur. We hypothesize that the use of polymer scaffolds could overcome these problems and support the establishment of three-dimensional (3D) culture systems in pharmaceutical research. Isolated primary rat hepatocytes were cultured on collagen-coated nanofibrous scaffolds for 7 days. Cell loading efficiency was quantified via DNA content measurement. Cell viability and presence of liver-cell-specific functions (albumin secretion, glycogen storage capacity) were evaluated. The activity of liver-specific factors was analyzed by immunofluorescent staining. RNA was isolated to establish quantitative real-time PCR. Our results indicate that primary rat hepatocytes cultured on nanofibrous scaffolds revealed high viability and well-preserved glycogen storage. Albumin secretion was existent during the entire culture period. Hepatocytes remain HNF-4 positive, indicating highly preserved cell differentiation. Aggregated hepatocytes re-established positive signaling for Connexin 32, a marker for differentiated hepatocyte interaction. ZO-1-positive hepatocytes were detected indicating formation of tight junctions. Expression of cytochrome isoenzymes was inducible. Altogether the data suggest that nanofibrous scaffolds provide a good in vitro microenvironment for neo tissue regeneration of primary rat hepatocytes.
原代大鼠肝细胞是一种广泛用于评估药物代谢和毒性的实验模型。在目前使用的二维（2D）细胞培养系统中，会出现典型的形态变化和肝细胞特异性功能丧失等问题。我们假设使用聚合物支架可以克服这些问题，并支持在药物研究中建立三维（3D）培养系统。分离的原代大鼠肝细胞在胶原涂覆的纳米纤维支架上培养 7 天。通过 DNA 含量测量来定量细胞加载效率。评估细胞活力和存在的肝细胞特异性功能（白蛋白分泌、糖原储存能力）。通过免疫荧光染色分析肝特异性因子的活性。分离 RNA 以建立定量实时 PCR。我们的结果表明，在纳米纤维支架上培养的原代大鼠肝细胞具有较高的活力和良好的糖原储存能力。白蛋白分泌在整个培养期间存在。肝细胞仍然为 HNF-4 阳性，表明细胞分化高度保留。聚集的肝细胞重新建立了用于分化的肝细胞相互作用的标志物 Connexin 32 的阳性信号。检测到 ZO-1 阳性的肝细胞，表明形成了紧密连接。细胞色素同工酶的表达具有诱导性。总的来说，这些数据表明纳米纤维支架为原代大鼠肝细胞的新组织再生提供了良好的体外微环境。
