Division of Experimental Surgery, Berlin Brandenburg Center for Regenerative Therapies (BCRT), Charité Universitätsmedizin Berlin, Campus Virchow-Klinikum, Augustenburger Platz 1, 13353 Berlin, Germany.
Biotechnol Bioeng. 2012 Dec;109(12):3172-81. doi: 10.1002/bit.24573. Epub 2012 Jun 27.
Based on a hollow fiber perfusion technology with internal oxygenation, a miniaturized bioreactor with a volume of 0.5 mL for in vitro studies was recently developed. Here, the suitability of this novel culture system for pharmacological studies was investigated, focusing on the model drug diclofenac. Primary human liver cells were cultivated in bioreactors and in conventional monolayer cultures in parallel over 10 days. From day 3 on, diclofenac was continuously applied at a therapeutic concentration (6.4 µM) for analysis of its metabolism. In addition, the activity and gene expression of the cytochrome P450 (CYP) isoforms CYP1A2, CYP2B6, CYP2C9, CYP2D6, and CYP3A4 were assessed. Diclofenac was metabolized in bioreactor cultures with an initial conversion rate of 230 ± 57 pmol/h/10(6) cells followed by a period of stable conversion of about 100 pmol/h/10(6) cells. All CYP activities tested were maintained until day 10 of bioreactor culture. The expression of corresponding mRNAs correlated well with the degree of preservation. Immunohistochemical characterization showed the formation of neo-tissue with expression of CYP2C9 and CYP3A4 and the drug transporters breast cancer resistance protein (BCRP) and multidrug resistance protein 2 (MRP2) in the bioreactor. In contrast, monolayer cultures showed a rapid decline of diclofenac conversion and cells had largely lost activity and mRNA expression of the assessed CYP isoforms at the end of the culture period. In conclusion, diclofenac metabolism, CYP activities and gene expression levels were considerably more stable in bioreactor cultures, making the novel bioreactor a useful tool for pharmacological or toxicological investigations requiring a highly physiological in vitro representation of the liver.
基于内部供氧的中空纤维灌注技术，最近开发了一种体积为 0.5 毫升的小型化生物反应器，用于体外研究。本文研究了该新型培养系统在药理学研究中的适用性，重点研究了模型药物双氯芬酸。原代人肝细胞在生物反应器中和传统单层培养物中平行培养 10 天。从第 3 天开始，以治疗浓度（6.4 µM）连续应用双氯芬酸以分析其代谢。此外，还评估了细胞色素 P450（CYP）同工酶 CYP1A2、CYP2B6、CYP2C9、CYP2D6 和 CYP3A4 的活性和基因表达。双氯芬酸在生物反应器培养物中代谢，初始转化率为 230±57 pmol/h/106 细胞，随后稳定转化率约为 100 pmol/h/106 细胞。测试的所有 CYP 活性均保持至生物反应器培养的第 10 天。相应的 mRNA 表达与保存程度密切相关。免疫组织化学特征显示在生物反应器中形成具有 CYP2C9 和 CYP3A4 表达的新组织以及乳腺癌耐药蛋白（BCRP）和多药耐药蛋白 2（MRP2）的药物转运蛋白。相比之下，单层培养物中双氯芬酸转化率迅速下降，在培养期末，评估的 CYP 同工酶的细胞活性和 mRNA 表达已大大丧失。总之，双氯芬酸代谢、CYP 活性和基因表达水平在生物反应器培养物中更为稳定，使新型生物反应器成为需要高度生理模拟肝脏的药理学或毒理学研究的有用工具。
