Baudoin Régis, Alberto Giulia, Legendre Audrey, Paullier Patrick, Naudot Marie, Fleury Marie-José, Jacques Sébastien, Griscom Laurent, Leclerc Eric
CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Université de Technologie de Compiègne, France.
Biotechnol Prog. 2014 Mar-Apr;30(2):401-10. doi: 10.1002/btpr.1857. Epub 2014 Jan 11.
We investigated the behavior of primary rat hepatocytes in biochips using a microfluidic platform (the integrated dynamic cell culture microchip). We studied the effects of cell inoculation densities (0.2-0.5 × 10(6) cells/biochip) and perfusion flow rates (10, 25, and 40 µL/min) during 72 h of perfusion. No effects were observed on hepatocyte morphology, but the levels of mRNA and CYP1A2 activity were found to be dependent on the initial cell densities and flow rates. The dataset made it possible to extract a best estimated range of parameters in which the rat hepatocytes appeared the most functional in the biochips. Namely, at 0.25 × 10(6) inoculated cells cultivated at 25 µL/min for 72 h, we demonstrated better induction of the expression of all the genes analyzed in comparison with other cell densities and flow rates. More precisely, when primary rat hepatocytes were cultivated at these conditions, the time-lapse analysis demonstrated an over expression of CYP3A1, CYP2B1, ABCC1b and ABCC2 in the biochips when compared to the postextraction levels. Furthermore, the AHR, CYP1A2, GSTA2, SULT1A1, and UGT1A6 levels remained higher than 50% of the postextraction values whereas values of HNF4α, CEBP, and PXR remained higher than 20% during the duration of the culture process. Nevertheless, an important reduction in mRNA levels was found for the xenosensors CAR and FXR, and the related CYP (CYP2E1, CYP7A1, CYP3A2, and CYP2D2). CYP1A2 functionality was illustrated by 700 ± 100 pmol/h/10(6) cells resorufin production. This study highlighted the functionality in optimized conditions of primary rat hepatocytes in parallelized microfluidic cultures and their potential for drug screening applications.
我们使用微流控平台（集成动态细胞培养微芯片）研究了原代大鼠肝细胞在生物芯片中的行为。我们研究了接种密度（0.2 - 0.5×10⁶个细胞/生物芯片）和灌注流速（10、25和40微升/分钟）在72小时灌注过程中的影响。未观察到对肝细胞形态的影响，但发现mRNA水平和CYP1A2活性取决于初始细胞密度和流速。该数据集使得能够提取出参数的最佳估计范围，在该范围内大鼠肝细胞在生物芯片中表现出最强的功能。具体而言，在以25微升/分钟的流速培养72小时的0.25×10⁶个接种细胞的情况下，与其他细胞密度和流速相比，我们证明了所分析的所有基因的表达诱导效果更好。更确切地说，当原代大鼠肝细胞在这些条件下培养时，延时分析表明，与提取后水平相比，生物芯片中CYP3A1、CYP2B1、ABCC1b和ABCC2出现过表达。此外，在培养过程中，AHR、CYP1A2、GSTA2、SULT1A1和UGT1A6水平保持高于提取后值的50%，而HNF4α、CEBP和PXR的值在培养过程中保持高于20%。然而，发现异生素传感器CAR和FXR以及相关的CYP（CYP2E1、CYP7A1、CYP3A2和CYP2D2）的mRNA水平有显著降低。CYP1A2的功能通过每10⁶个细胞每小时产生700±100皮摩尔试卤灵来体现。这项研究突出了原代大鼠肝细胞在平行微流控培养优化条件下的功能及其在药物筛选应用中的潜力。
