title: Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips.
abstract: 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.
authors.affiliation: CNRS UMR 7338, Laboratoire de Biomécanique et Bioingénierie, Université de Technologie de Compiègne, France.
authors.forename: Régis
authors.initials: R
authors.lastname: Baudoin
authors.forename: Giulia
authors.initials: G
authors.lastname: Alberto
authors.forename: Audrey
authors.initials: A
authors.lastname: Legendre
authors.forename: Patrick
authors.initials: P
authors.lastname: Paullier
authors.forename: Marie
authors.initials: M
authors.lastname: Naudot
authors.forename: Marie-José
authors.initials: MJ
authors.lastname: Fleury
authors.forename: Sébastien
authors.initials: S
authors.lastname: Jacques
authors.forename: Laurent
authors.initials: L
authors.lastname: Griscom
authors.forename: Eric
authors.initials: E
authors.lastname: Leclerc
mesh_terms: D000818:Animals; D002470:Cell Survival; D002478:Cells, Cultured; D003577:Cytochrome P-450 Enzyme System / Q000235:genetics / Q000378:metabolism*; D004867:Equipment Design; D020869:Gene Expression Profiling / Q000379:methods*; D022781:Hepatocytes / Q000737:chemistry / Q000378:metabolism*; D046210:Microfluidic Analytical Techniques / Q000379:methods*; D012333:RNA, Messenger / Q000235:genetics / Q000378:metabolism*; D051381:Rats
chemical_list: D012333:RNA, Messenger; D003577:Cytochrome P-450 Enzyme System
keywords: IDCCM; density effect; flow rate effect; gene expressions; microfluidic biochips; primary rat hepatocytes