Nanotechnologies et ingénierie du foie bioartificiel. Une autre idée de la « convergence technologique »

La conception américaine de la convergence technologique, promue par le programme NBIC (Nano-Bio-Info-Cogno), est orientée vers la recherche d’une intégration de tous les domaines du réel (la nature, la vie, l’esprit, la société) dans la représentation unitaire d’un monde constitué de systèmes hiérarchiques complexes et couplés entre eux. Il s’agit par conséquent d’une vision totalisante, témoignant d’un idéal de contrôle et de maîtrise que rien ne paraît devoir limiter. Les Grecs avaient un ..

Human hepatic cell behavior on polysulfone membrane with double porosity level

In the membrane-based bioartificial livers developed up to now,the hepatic cells were located either in the fibers lumen or in the cartridge,with limited capacity for cell hosting.Here,we designed a polysulfone (PSU) membrane with a double porosity level:(i)surface macroporosity emerging in macrochambers accessible to hepatic cell colonization;(ii)microporosity to ensure gas and molecule transfers between macrochambers and supernatant,as well as potential immune barrier. ESEM and X-ray tomography confirmed that macrochambers accessed the membrane surface and were inter-connected. Biocompatibility and performances of this PSU membrane with double porosity level were compared to classical semi-permeable structures,following cell organization,cell proliferation and liver specific activities over a 9 days incubation. Macrochambers were colonized by hepatic cells, leading to higher albumin synthesis compared to control.Therefore,this membrane with double porosity appeared as a promising support to offer an inner 3D environment adequate to cell proliferation to form a liver-like tissue

Encapsulation of Huh-7 cells within alginate-poly(ethylene glycol) hybrid microspheres

Novel calcium alginate poly(ethylene glycol) hybrid microspheres (Ca-alg-PEG) were developed and evaluated as potentially suitable materials for cell microencapsulation. Grafting 5-13% of the backbone units of sodium alginate (Na-alg) with α-amine-ω-thiol PEG maintained the gelling capacity in presence of calcium ions, while thiol end groups allowed for preparing chemically crosslinked hydrogel via spontaneous disulfide bond formation. The combination of these two gelling mechanisms yielded Ca-alg-PEG. Human hepatocellular carcinoma cells (Huh-7) were encapsulated in Ca-alg-PEG and calcium alginate beads (Ca-alg), and cultured for 2weeks under agitation conditions. Immediately after completion of the microencapsulation, the cell viability was 60% and similar in Ca-alg-PEG and Ca-alg. The proliferation of Huh-7 encapsulated in Ca-alg-PEG was slightly higher than in Ca-alg. Accelerated proliferation after 2weeks was observed for the encapsulation in Ca-alg-PEG. The production of albumin confirmed the functionality of the encapsulated Huh-7 cells. The study confirms the suitability of Ca-alg-PEG and the one-step technology for cell microencapsulatio

Numerical study of the influence of wall compliance on the hemodynamics in a patient-specific arteriovenous fistula

An arteriovenous fistula (AVF) is a vessel connection created to provide adequate blood access for hemodialysis. The local hemodynamics was investigated in a patient-specific AVF using a computational fluid structure interaction simulation. The fluid and solid governing equations were solved using ANSYS (coupling CFX and ANSYS Structural) imposing physiological boundary conditions and vessel mechanical properties. An accurate map of unsteady velocity profiles and wall shear stresses was drawn up. It was compared to rigid wall simulations to quantify the effect of the wall compliance

A new prescription model for regional citrate anticoagulation in therapeutic plasma exchanges

Regional citrate anticoagulation (RCA) is proposed for various extracorporeal purification techniques to overcome the risk of bleeding that might result from systemic anticoagulation. Yet, no individualized treatment protocol has been proposed for therapeutic plasma exchange (TPE) so far. The objective of this study was to assess the determinants of blood citrate concentration needed and to develop an individualized RCA protocol useful for clinical practice. The study population included 14 patients who underwent a total of 47 TPE sessions. Citrate was infused pre-plasmafilter. Post-plasmafilter and systemic plasma ionized calcium concentrations were measured at standardized time intervals. An algorithm was proposed for the supplementation of calcium. During the discovery phase, citrate was infused at a fixed starting rate, and adapted accordingly to obtained post-plasmafilter ionized calcium levels. Using a mathematical approach, an algorithm was thereafter developed for individualized prescriptions of citrate. Pre-treatment values of hematocrit and plasma ionized calcium were the main determinants of the required rate of citrate infusion. These can be integrated into a final equation enabling to individualize the prescription. A prefilter ionized calcium concentration between 0.24 and 0.33 mmol/l prevented coagulation of the extracorporeal circuit. Significant hypocalcemia occurred in 8.5% of treatments. There were no significant acid-base disturbances. We propose a new protocol, which enables for the first time to individualize the prescription of regional citrate anticoagulation during TPE, in an efficient manner. The immediately obtained regional anticoagulation protects against both the risk of coagulation of the membrane and the exposure to an excess of citrate

Expansion properties of Alginate beads as cell carrier in the fluidized bed bioartificial liver

The homogeneous expansion behaviour of liquid-fluidized beds is exploited in various fields such as minerals engineering and biotechnology. Innovative fluidized bed bioreactor concepts have been also explored for applications as bioartificial organs, particularly the bioartificial liver (1). It has been shown that the fluidized bed bioreactor constituted of alginate beads hosting liver cells is one of the promising solution to a bioartificial liver. Compared to other solutions, fluidization of alginate beads containing the cells does not suffer from the severe limitations to mass transfer between the beads and the perfusion medium. In the present work, appropriate alginate beads were prepared by the alginate drop gelation in calcium chloride. The beads were characterized in terms of size distribution and density. Sauter mean diameter of 813 m and density of 1020 kg/m3 were obtained. The latter shows a value very close to usual perfusion fluid, which required also careful evaluation of the liquid properties. Expansion properties were evaluated for free alginate beads (i.e. without hepatic cells) using saline solutions as fluidization medium. Bed expansions have been conducted in a small-size 1-cm diameter column used for perfusion in in vitro experiments as well as in a bigger 10-cm diameter column close to human size bioreactor. Velocity-voidage plots are reported and elaborated in terms of Richardson-Zaki parameters, showing the effect of walls and the different distributor. ACKNOLEDGEMENTS The financial support of the European Union through the Project FP7-PEOPLE-2012-ITN “Training network for developing innovative bioartificial devices for treatment of kidney and liver disease” is gratefully acknowledged. REFERENCES Gautier A., Carpentier B., Dufresne M., Vu Dinh Q., Paullier P., Legallais C. Impact of alginate type and bead diameter on mass transfers and the metabolic activities of encapsulated C3A cells in bioartificial liver applications. European Cells and Materials 2011, 21:94-106

Validation of HepG2/C3A Cell Cultures in Cyclic Olefin Copolymer Based Microfluidic Bioreactors

Organ-on-chip (OoC) technology is one of the most promising in vitro tools to replace the traditional animal experiment-based paradigms of risk assessment. However, the use of OoC in drug discovery and toxicity studies remain still limited by the low capacity for high-throughput production and the incompatibility with standard laboratory equipment. Moreover, polydimethylsiloxanes, the material of choice for OoC, has several drawbacks, particularly the high absorption of drugs and chemicals. In this work, we report the development of a microfluidic device, using a process adapted for mass production, to culture liver cell line in dynamic conditions. The device, made of cyclic olefin copolymers, was manufactured by injection moulding and integrates Luer lock connectors compatible with standard medical and laboratory instruments. Then, the COC device was used for culturing HepG2/C3a cells. The functionality and behaviour of cultures were assessed by albumin secretion, cell proliferation, viability and actin cytoskeleton development. The cells in COC device proliferated well and remained functional for 9 days of culture. Furthermore, HepG2/C3a cells in the COC biochips showed similar behaviour to cells in PDMS biochips. The present study provides a proof-of-concept for the use of COC biochip in liver cells culture and illustrate their potential to develop OoC.This research was funded by Basque Country Government within the frame of the project BIKAINTEK 2018 (48-AF-W2-2018-00006). Taha Messelmani PhD is funded by ANR (Agence National de la Recherche, France, MIMLIVEROnChip ANR-19-CE19-0020-01 project)

Predictive toxicology using systemic biology and liver microfluidic "on chip" approaches: Application to acetaminophen injury

International audienceWe have analyzed transcriptomic, proteomic and metabolomic profiles of hepatoma cells cultivated inside a microfluidic biochip with or without acetaminophen (APAP). Without APAP, the results show an adaptive cellular response to the microfluidic environment, leading to the induction of anti-oxidative stress and cytoprotective pathways. In presence of APAP, calcium homeostasis perturbation, lipid peroxidation and cell death are observed. These effects can be attributed to APAP metabolism into its highly reactive metabolite. N-acetyl-p-benzoquinone imine (NAPQI). That toxicity pathway was confirmed by the detection of GSH-APAP, the large production of 2-hydroxybutyrate and 3-hydroxybutyrate, and methionine, cystine, and histidine consumption in the treated biochips. Those metabolites have been reported as specific biomarkers of hepatotoxicity and glutathione depletion in the literature. In addition, the integration of the metabolomic, transcriptomic and proteomic collected profiles allowed a more complete reconstruction of the APAP injury pathways. To our knowledge, this work is the first example of a global integration of microfluidic biochip data in toxicity assessment. Our results demonstrate the potential of that new approach to predictive toxicology

Integrated Proteomic and Transcriptomic Investigation of the Acetaminophen Toxicity in Liver Microfluidic Biochip

Microfluidic bioartificial organs allow the reproduction of in vivo-like properties such as cell culture in a 3D dynamical micro environment. In this work, we established a method and a protocol for performing a toxicogenomic analysis of HepG2/C3A cultivated in a microfluidic biochip. Transcriptomic and proteomic analyses have shown the induction of the NRF2 pathway and the related drug metabolism pathways when the HepG2/C3A cells were cultivated in the biochip. The induction of those pathways in the biochip enhanced the metabolism of the N-acetyl-p-aminophenol drug (acetaminophen-APAP) when compared to Petri cultures. Thus, we observed 50% growth inhibition of cell proliferation at 1 mM in the biochip, which appeared similar to human plasmatic toxic concentrations reported at 2 mM. The metabolic signature of APAP toxicity in the biochip showed similar biomarkers as those reported in vivo, such as the calcium homeostasis, lipid metabolism and reorganization of the cytoskeleton, at the transcriptome and proteome levels (which was not the case in Petri dishes). These results demonstrate a specific molecular signature for acetaminophen at transcriptomic and proteomic levels closed to situations found in vivo. Interestingly, a common component of the signature of the APAP molecule was identified in Petri and biochip cultures via the perturbations of the DNA replication and cell cycle. These findings provide an important insight into the use of microfluidic biochips as new tools in biomarker research in pharmaceutical drug studies and predictive toxicity investigations

Mise en place et validation d'un modèle in vitro pour l'étude des propriétés mécaniques, diffusives et métaboliques d'un foie bioartificiel à lit fluidisé

COMPIEGNE-BU (601592101) / SudocSudocFranceF