Cécile Legallais

Blood flow patterns in an anatomically realistic coronary vessel: influence of three different reconstruction methods

Many clinical studies suggest that local blood flow patterns are involved in the location and development of atherosclerosis. In coronary diseases, this assumption should be corroborated by quantitative information on local hemodynamic parameters such as pressure, velocity or wall shear stress. Nowadays, computational fluid dynamics (CFD) algorithms coupled to realistic 3-D reconstructions of such vessels make these data accessible. Nevertheless, they should be carefully analysed to avoid misinterpretations when the physiological parameters are not all considered. As an example, we propose here to compare the flow patterns calculated in a coronary vessel reconstructed by three different methods. In the three cases, the vessel trajectory respected the physiology. In the simplest reconstruction, the coronary was modelled by a tube of constant diameter while in the most complex one, the cross-sections corresponded to the reality. We showed that local pressures, wall shear rates and velocity profiles were severely affected by the geometrical modifications. In the constant cross-section vessel, the flow resembled to that of Poiseuille in a straight tube. On the contrary, velocity and shear rate exhibited sudden local variations in the more realistic vessels. As an example, velocity could be multiplied by 5 as compared to Poiseuilles flow and area of very low wall shear rates appeared. The results obtained with the most complex model clearly outlined that, in addition to a proper description of the vessel trajectory, the section area changes should be carefully taken into account, confirming assumptions already highlighted before the rise of commercially available and efficient CFD softwares.

Improvement of HepG2/C3a cell functions in a microfluidic biochip

Current developments in tissue engineering and microtechnology fields allow the use of microfluidic biochip as microtools for in vitro investigations. In the present study, we describe the behavior of HepG2/C3a cells cultivated in a poly(dimethylsiloxane) (PDMS) microfluidic biochip coupled to a perfusion system. Cell culture in the microfluidic biochip for 96 h including 72 h of perfusion provoked a 24 h delay in cell growth compared to plate cultures. Inside the microfluidic biochip, few apoptosis, and necrosis were detected along the culture and 3D cell organization was observed. Regarding the hepatic metabolism, glucose and glutamine consumptions as well as albumin synthesis were maintained. A transcriptomic analysis performed at 96 h of culture using Affymetrix GeneChip demonstrated that 1,025 genes with a fold change above 1.8 were statistically differentially expressed in the microfluidic biochip cultures compared to plate cultures. Among those genes, phase I enzymes involved in the xenobiotics metabolism such as the cytochromes P450 (CYP) 1A1/2, 2B6, 3A4, 3A5, and 3A7 were up‐regulated. The CYP1A1/2 up‐regulation was associated with the appearance of CYP1A1/2s activity evidenced by using EROD biotransformation assay. Several phase II enzymes such as sulfotransferases (SULT1A1 and SULT1A2), UDP‐glucuronyltransferase (UGT1A1, UGT2B7) and phase III transporters (such as MDR1, MRP2) were also up‐regulated. In conclusion, microfluidic biochip could and provide an important insight to exploring the xenobiotics metabolism. Altogether, these results suggest that this kind of biochip could be considered as a new pertinent tool for predicting cell toxicity and clearance of xenobiotics in vitro. Biotechnol. Bioeng. 2011; 108:1704–1715.

Bioartificial livers (BAL): current technological aspects and future developments

Abstract Several attempts have been made to develop bioartificial support for the treatment of patients with acute or fulminant liver failure. In this paper, the authors have reviewed the technological aspects of the previously developed bioreactor devices like membrane-based devices, direct perfusion systems and entrapment-based columns. For each type, the technological requirements were first theoretically addressed and then confronted with the actual bioreactor configurations. A new bioreactor design has been proposed to enhance the performance of a bioartificial liver consisting of porcine hepatocytes entrapped in alginate beads. Finally, perspectives were drawn for the next developments of such promising bioartificial organs.

Alginate-encapsulated HepG2 Cells in a Fluidized Bed Bioreactor Maintain Function in Human Liver Failure Plasma

Alginate-encapsulated HepG2 cells cultured in microgravity have the potential to serve as the cellular component of a bioartificial liver. This study investigates their performance in normal and liver failure (LF) human plasma over 6-8 h in a fluidized bed bioreactor. After 8 days of microgravity culture, beads containing 1.5 x 10(9) cells were perfused for up to 8 h at 48 mL/min with 300 mL of plasma. After exposure to 90% LF plasma, vital dye staining showed maintained cell viability, while a 7% increase in lactate dehydrogenase activity indicated minimal cell damage. Glucose consumption, lactate production, and a 4.3-fold linear increase in alpha-fetoprotein levels were observed. Detoxificatory function was demonstrated by quantification of bilirubin conjugation, urea synthesis, and Cyp450 1A activity. These data show that in LF plasma, alginate-encapsulated HepG2 cells can maintain viability, and metabolic, synthetic, and detoxificatory activities, indicating that the system can be scaled-up to form the biological component of a bioartificial liver.

Investigation of ifosfamide nephrotoxicity induced in a liver-kidney co-culture biochip.

In this article, we present a liver–kidney co‐culture model in a micro fluidic biochip. The liver was modeled using HepG2/C3a and HepaRG cell lines and the kidney using MDCK cell lines. To demonstrate the synergic interaction between both organs, we investigated the effect of ifosfamide, an anticancerous drug. Ifosfamide is a prodrug which is metabolized by the liver to isophosforamide mustard, an active metabolite. This metabolism process also leads to the formation of chloroacetaldehyde, a nephrotoxic metabolite and acrolein a urotoxic one. In the biochips of MDCK cultures, we did not detect any nephrotoxic effects after 72 h of 50 µM ifosfamide exposure. However, in the liver–kidney biochips, the same 72 h exposure leads to a nephrotoxicity illustrated by a reduction of the number of MDCK cells (up to 30% in the HepaRG‐MDCK) when compared to untreated co‐cultures or treated MDCK monocultures. The reduction of the MDCK cell number was not related to a modification of the cell cycle repartition in ifosfamide treated cases when compared to controls. The ifosfamide biotransformation into 3‐dechloroethylifosfamide, an equimolar byproduct of the chloroacetaldehyde production, was detected by mass spectrometry at a rate of apparition of 0.3 ± 0.1 and 1.1 ± 0.3 pg/h/biochips in HepaRG monocultures and HepaRG‐MDCK co‐cultures respectively. Any metabolite was detected in HepG2/C3a cultures. Furthermore, the ifosfamide treatment in HepaRG‐MDCK co‐culture system triggered an increase in the intracellular calcium release in MDCK cells on contrary to the treatment on MDCK monocultures. As 3‐dechloroethylifosfamide is not toxic, we have tested the effect of equimolar choloroacetaldehyde concentration onto the MDCK cells. At this concentration, we found a quite similar calcium perturbation and MDCK nephrotoxicity via a reduction of 30% of final cell numbers such as in the ifosfamide HepaRG‐MDCK co‐culture experiments. Our results suggest that ifosfamide nephrotoxicity in a liver–kidney micro fluidic co‐culture model using HepaRG‐MDCK cells is induced by the metabolism of ifosfamide into chloroacetaldehyde whereas this pathway is not functional in HepG2/C3a‐MDCK model. This study demonstrates the interest in the development of systemic organ–organ interactions using micro fluidic biochips. It also illustrated their potential in future predictive toxicity model using in vitro models as alternative methods. Biotechnol. Bioeng. 2013; 110: 597–608.

In Vitro Assessment of Encapsulated C3A Hepatocytes Functions in a Fluidized Bed Bioreactor

In the present in vitro model, the authors intended to assess viability and functionality of hepatocytes encapsulated into alginate beads and submitted to a fluidized bed motion in a bioreactor. Human immortalized C3A line was chosen as cell model. Two controls consisting of (1) cells cultured on flasks and (2) cells encapsulated in alginate beads under static conditions were implemented. The cell functions studied were total protein, albumin, urea, and ammonia synthesis, as well as ammonia removal in the case of overdose. The comparison among the three cases studied showed that the three‐dimensional structure of alginate offered a suitable environment for cell functions. In addition, the fluidized bed bioreactor enhanced the mass transfer and thus increased the amount of species released out of the beads, as compared with the static case. Ammonia detoxification only appeared reduced by encapsulation. The concept of a fluidized bed bioartificial liver was thus validated by this in vitro model, which indicated that cell functions could be efficiently retained. In addition, as far as urea and protein synthesis and release were concerned, the use of the C3A cell line, in combination with encapsulation and fluidization technology, offered a real potentiality for the purpose of extracorporeal liver supply.

Experimental kinetic aspects of hollow fiber membrane-based pseudobioaffinity filtration: process for IgG separation from human plasma

The pseudobiospecific affinity ligand l-histidine was immobilized through an ether linkage onto poly(ethylene vinyl alcohol) hollow fiber cartridge to obtain an affinity support for IgG purification or IgG removal from human plasma with a view to clinical apheresis for the treatment of immune disorders, alloimmunization and cancer. The kinetics and mass transfer aspects were studied in order to determine the optimum loading flow rate for IgG adsorption. Experiments were run at various filtrate flow rates (various residence times) in cross flow mode with recirculation of both the retentate and the permeate in the reservoir. The results showed that this affinity membrane had the potential of fractionating plasma proteins at fast flow rates (residence times below 10 s). However, protein trapping into the membrane was enhanced by residence times below 5 s. Comparison of equilibrium and dynamic maximum capacities determined using Langmuir isotherms showed that dynamic capacity (190 μg/cm2) was somewhat higher than the equilibrium capacity (148 μg/cm2). The dissociation constants for IgG were determined using the Langmuir isotherm equation to be 3.7 × 10−6 M (dynamic mode) and 9.8 × 10−6 M (equilibrium binding analysis), indicating medium affinity, which was typical for pseudobiospecific affinity ligands. The cartridge was loaded with human serum or human plasma to test its ability to fractionate these protein solutions. The affinity membrane showed high selectivity for IgG from two fold diluted human plasma or serum. IgM was also adsorbed to a certain extent. When the serum or plasma was not diluted with equilibration buffer before loading the cartridge, the amount of adsorbed proteins was significantly (50%) smaller than with the 2 or 10 fold dilutions, but the specificity remained.

Analysis of transcriptomic and proteomic profiles demonstrates improved Madin–Darby canine kidney cell function in a renal microfluidic biochip

We have evaluated the influence of the microfluidic environment on renal cell functionality. For that purpose, we performed a time lapse transcriptomic and proteomic analysis in which we compared gene and protein expressions of Madin–Darby canine kidney cells after 24 h and 96 h of culture in both microfluidic biochips and plates. The transcriptomic and proteomic integration revealed that the ion transporters involved in calcium, phosphate, and sodium homoeostasis and several genes involved in H+ transporters and pH regulation were up‐regulated in microfluidic biochips. Concerning drug metabolism, we found Phase I (CYP P450), Phase II enzymes (GST), various multidrug resistance genes (MRP), and Phase III transporters (SLC) were also up‐regulated in the biochips. Furthermore, the study shows that those inductions were correlated with the induction of the Ahr and Nrf‐2 dependent pathways, which results in a global cytoprotective response induced by the microenvironment. However, there was no apoptosis situation or cell death in the biochips. Microfluidic biochips may thus provide an important insight into exploring xenobiotic injury and transport modifications in this type of bioartificial microfluidic kidney. Finally, the investigation demonstrated that combining the transcriptomic and proteomic analyses obtained from a cell “on chip” culture would provide a pertinent new tool in the mechanistic interpretation of cellular mechanisms for predicting kidney cell toxicity and renal clearance in vitro.

A potential set up based on histidine hollow fiber membranes for the extracorporeal removal of human antibodies

Abstract Poly ethylene vinyl alcohol (PEVA) based hollow fiber membrane grafted with l -histidine has been found to be particularly useful for the preparative scale separation of immunoglobulin G and potential clinical applications. In this paper, we present a set-up designed for the extracorporeal removal of immunoglobulin G. Biochemical and hydrodynamic conditions were optimized so as to perform in vitro removal of 50% of plasma immunoglobulin G.

Bioengineering the Liver: Scale-Up and Cool Chain Delivery of the Liver Cell Biomass for Clinical Targeting in a Bioartificial Liver Support System

Abstract Acute liver failure has a high mortality unless patients receive a liver transplant; however, there are insufficient donor organs to meet the clinical need. The liver may rapidly recover from acute injury by hepatic cell regeneration given time. A bioartificial liver machine can provide temporary liver support to enable such regeneration to occur. We developed a bioartificial liver machine using human-derived liver cells encapsulated in alginate, cultured in a fluidized bed bioreactor to a level of function suitable for clinical use (performance competence). HepG2 cells were encapsulated in alginate using a JetCutter to produce ∼500 μm spherical beads containing cells at ∼1.75 million cells/mL beads. Within the beads, encapsulated cells proliferated to form compact cell spheroids (AELS) with good cell-to-cell contact and cell function, that were analyzed functionally and by gene expression at mRNA and protein levels. We established a methodology to enable a ∼34-fold increase in cell density within the AELS over 11–13 days, maintaining cell viability. Optimized nutrient and oxygen provision were numerically modeled and tested experimentally, achieving a cell density at harvest of >45 million cells/mL beads; >5×1010 cells were produced in 1100 mL of beads. This process is scalable to human size ([0.7–1]×1011). A short-term storage protocol at ambient temperature was established, enabling transport from laboratory to bedside over 48 h, appropriate for clinical translation of a manufactured bioartificial liver machine.