Metabolic Characterization of Primary Rat Hepatocytes Cultivated in Parallel Microfluidic Biochips

Legendre, Audrey; Baudoin, R.; Alberto, Giulia; Paullier, Patrick; Naudot, Marie; Bricks, Thibault; Brocheton, Jessy; Jacques, Sébastien; Cotton, J. P.; Leclerc, Éric

doi:10.1002/jps.23466

Cited by 50 publications

(33 citation statements)

References 49 publications

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“…4 Furthermore, different microfluidic systems have been developed in the past in which constant growth conditions were achieved by a perfusion flow of cell culture medium providing permanent sustenance with nutrients and oxygen as well as removal of waste metabolites. 18,19 However, to our knowledge, there is still no system described where hepatocytes are cultivated with indirect flow without any physical barrier which would reflect the in vivo situation even better. In order to establish a useful cultivation system for the analysis of hepatocellular functions, we tested the growth, differentiation, and metabolical behavior of HepG2 cells embedded in Matrigel in the OrganoPlate from MIMETAS and Leiden University that combines these unique characteristics.…”

Section: Discussionmentioning

confidence: 99%

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

et al. 2015

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The in vitro study of liver functions and liver cell specific responses to external stimuli deals with the problem to preserve the in vivo functions of primary hepatocytes. In this study, we used the biochip OrganoPlate TM (MIMETAS) that combines different advantages for the cultivation of hepatocytes in vitro: (1) the perfusion flow is achieved without a pump allowing easy handling and placement in the incubator; (2) the phaseguides allow plating of matrix-embedded cells in lanes adjacent to the perfusion flow without physical barrier; and (3) the matrix-embedding ensures indirect contact of the cells to the flow. In order to evaluate the applicability of this biochip for the study of hepatocyte's functions, Matrigel TM -embedded HepG2 cells were cultured over three weeks in this biochip and compared to a static Matrigel culture (3D) and a monolayer culture (2D). Chip-cultured cells grew in spheroid-like structures and were characterized by the formation of bile canaliculi and a high viability over 14 days. Hepatocyte-specific physiology was achieved as determined by an increase in albumin production. Improved detoxification metabolism was demonstrated by strongly increased cytochrome P450 activity and urea production. Additionally, chip-cultured cells displayed increased sensitivity to acetaminophen. Altogether, the OrganoPlate seems to be a very useful alternative for the cultivation of hepatocytes, as their behavior was strongly improved over 2D and static 3D cultures and the results were largely comparable and partly superior to the previous reports on biochip-cultured hepatocytes. As for the low technical needs, this platform has the appearance of being highly applicable for further studies of hepatocytes' responses to external stimuli. V C 2015 AIP Publishing LLC. [http://dx

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Section: Discussionmentioning

confidence: 99%

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

et al. 2015

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“…The feasibility of carrying out transcriptomic, proteomic, metabolic and toxicologic characterization with this arrangement, was illustrated and showed that HepG2/C3a phenotypic responses including albumin secretion, sensitivity to acetaminophen toxicity, and relative expression of CYP450 and transport genes appeared more physiologically close to primary hepatocytes in the microfluidic compared to static petri dish culture, a result that may in part due to the quasi-3D aggregate structures that form in regions of the recessed structures [74,79,80][81–84]. Initial studies analyzing primary rat [85,86] and human [74,87] cells in short term culture (4 days) indicate that these cells were less likely to form aggregates, and optimization of operating conditions for these cells is evolving. The results assessing drug metabolism with cryopreserved human cells are instructive in two key ways, first showing that these cells attach and survive in both the recessed features as well as the top of the PDMS exposed directly to flow.…”

Section: Bioreactorsmentioning

confidence: 99%

Bioreactor technologies to support liver function in vitro

Ebrahimkhani

Neiman

Raredon

et al. 2014

Advanced Drug Delivery Reviews

119

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Liver is a central nexus integrating metabolic and immunologic homeostasis in the human body, and the direct or indirect target of most molecular therapeutics. A wide spectrum of therapeutic and technological needs drive efforts to capture liver physiology and pathophysiology in vitro, ranging from prediction of metabolism and toxicity of small molecule drugs, to understanding off-target effects of proteins, nucleic acid therapies, and targeted therapeutics, to serving as disease models for drug development. Here we provide perspective on the evolving landscape of bioreactor-based models to meet old and new challenges in drug discovery and development, emphasizing design challenges in maintaining long-term liver-specific function and how emerging technologies in biomaterials and microdevices are providing new experimental models.

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“…Primary hepatocytes extraction protocol is introduced in detail in supplementary file 1 and in our previous works (Legendre et al, 2013). For the first 24 h, the freshly isolated hepatocytes were cultured in a seeding medium composed of William's E GLutamax medium (Fisher Scientific, Illkirch, France) supplemented with bovine insulin (5 mg/mL, Sigma-Aldrich, Saint-Quentin Fallavier, France) and bovine fetal serum (10%).…”

Section: Primary Rat Hepatocytesmentioning

confidence: 99%

“…Recently, miniaturization has helped to reduce this constraint. Several types of microscale tissues were proposed to perform "on chip" investigations such as with liver cells (Baudoin et al, 2014;Legendre et al, 2013;Novik et al, 2010), intestinal cells (Imura et al, 2009), kidney cells (Choucha et al, 2012;Ramello et al, 2011), or lung cells (Huh et al, 2010). Refined analyses involving drug clearances and the related toxicokinetics on targeted tissues can thus be proposed using co-culture devices.…”

Section: Introductionmentioning

confidence: 99%

First pass intestinal and liver metabolism of paracetamol in a microfluidic platform coupled with a mathematical modeling as a means of evaluating ADME processes in humans

Prot

Maciel

Bricks

et al. 2014

Biotech & Bioengineering

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We developed a microfluidic platform to investigate paracetamol intestinal and liver first pass metabolism. This approach was coupled with a mathematical model to estimate intrinsic in vitro parameters and to predict in vivo processes. The kinetic modeling estimated the paracetamol and paracetamol sulfate permeabilities, the sulfate and glucuronide effluxes in the intestine compartment. Based on a gut model, we estimated intrinsic intestinal clearance of between 26 and 77 L/h for paracetamol in humans, a permeability of 10 L/h, and a gut availability between 0.17 and 0.53 (compared to 0.95-1 in vivo). The role played by the liver in paracetamol metabolism was estimated via in vitro intrinsic clearances of 7.6, 13.6, and 11.5 µL/min/10(6) cells for HepG2/C3a, rat primary hepatocytes, and human primary hepatocytes, respectively. Based on a parallel tube model to describe the liver, the paracetamol hepatic clearance, and the paracetamol hepatic availability in humans were estimated at 6.5 mL/min/kg of bodyweight (BDW) and 0.7, respectively (when compared to 5 mL/min/kg of BDW and 0.77 to 0.88 for in vivo values, respectively). The drug availability was predicted ranging between 0.24 and 0.41 (0.88 in vivo). The overall approach provided a first step in an integrated strategy combining in silico/in vitro methods based on microfluidic for evaluating drug absorption, distribution and metabolism processes.

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Metabolic Characterization of Primary Rat Hepatocytes Cultivated in Parallel Microfluidic Biochips

Cited by 50 publications

References 49 publications

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

On-chip three-dimensional cell culture in phaseguides improves hepatocyte functions in vitro

Bioreactor technologies to support liver function in vitro

First pass intestinal and liver metabolism of paracetamol in a microfluidic platform coupled with a mathematical modeling as a means of evaluating ADME processes in humans

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