We used the recently introduced "metabolomics-on-a-chip" approach to test secondary drug toxicity in bioartificial organs. Bioartificial organs cultivated in microfluidic culture conditions provide a beneficial environment, in which the cellular cytoprotective mechanisms are enhanced, compared with Petri dish culture conditions. We investigated the metabolic response of HepG2/C3a cells exposed to flutamide, an anticancer prodrug, and hydroxyflutamide (HF), its active metabolite, in a microfluidic biochip. The cellular response was analyzed by (1)H nuclear magnetic resonance spectroscopy to identify cell-specific molecule-response markers. The metabolic response to flutamide results in a disruption of glucose homeostasis and in mitochondrial dysfunctions. This flutamide-specific metabolic response was illustrated by a reduction of the extracellular glucose and fructose consumptions and a general reduction of the tricarboxylic acid cycle activity leading to the reduction of the consumption of several amino acids. We also found a higher production of 3-hydroxybutyrate and lactate, and the reduction of the albumin production compared with controls. The toxic metabolic signature associated with the active metabolite HF was illustrated by a high-energy demand and an increase in several amino acid metabolism. Finally, for both molecules, the hepatotoxicity was correlated to the glutathione (GSH) metabolism illustrated by the levels of the 2-hydroxybutyrate and pyroglutamate productions and the increase of the glutamate and glycine productions. Thus, the entire set of results contributed to extract specific mechanistic toxic signatures and their relation to hepatotoxicity, which appeared consistent with literature reports. As new finding of HepG2/C3a cells hepatotoxicity, we propose a metabolic network with a related list of metabolite variations to describe the GSH depletion when followed by a cell death for the HepG2/C3a cells cultivated in our polydimethylsiloxane microfluidic biochips. Our findings illustrate the potential of metabolomics-on-a-chip as an in vitro alternative method for predictive toxicology.
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.
We have integrated in vitro and in silico information to investigate acetaminophen (APAP) and its metabolite N-acetyl-p-benzoquinone imine (NAPQI) toxicity in liver biochip. In previous works, we observed higher cytotoxicity of HepG2/C3a cultivated in biochips when exposed to 1 mM of APAP for 72 h as compared to Petri cultures. We complete our investigation with the present in silico approach to extend the mechanistic interpretation of the intracellular kinetics of the toxicity process. For that purpose, we propose a mathematical model based on the coupling of a drug pharmacokinetic model (PK) with a systemic biology model (SB) describing the reactive oxygen species (ROS) production by NAPQI and the subsequent glutathione (GSH) depletion. The SB model was parameterized using (i) transcriptomic data, (ii) qualitative results of time lapses ROS fluorescent curves for both control and 1-mM APAP-treated experiments, and (iii) additional GSH literature data. The PK model was parameterized (i) using the in vitro kinetic data (at 160 μM, 1 mM, 10 mM), (ii) using the parameters resulting from a physiologically based pharmacokinetic (PBPK) literature model for APAP, and (iii) by literature data describing NAPQI formation. The PK-SB model predicted a ROS increase and GSH depletion due to the NAPQI formation. The transition from a detoxification phase and NAPQI and ROS accumulation was predicted for a NAPQI concentration ranging between 0.025 and 0.25 μM in the cytosol. In parallel, we performed a dose response analysis in biochips that shows a reduction of the final hepatic cell number appeared in agreement with the time and doses associated with the switch of the NAPQI detoxification/accumulation. As a result, we were able to correlate in vitro extracellular APAP exposures with an intracellular in silico ROS accumulation using an integration of a coupled mathematical and experimental liver on chip approach.
Bone tissue engineering goes beyond the limitations of conventional methods of treating bone loss, such as autograft-induced morbidity and a lack of integration for large grafts. Novel biomimicry approaches (using three-dimensional [3D] electrospinning and printing techniques) have been designed to offer the most appro
Background Novel, less toxic, cost-effective and safe therapeutic strategies are needed to improve treatment of chronic lymphocytic leukemia (CLL). Ascorbic acid (AA, vitamin C) has shown a potential anti-cancer therapeutic activity in several cancers. However, the anti-cancer effects of ascorbic acid on CLL B-cells have not been extensively studied. We aimed in this study to evaluate the in vitro therapeutic activity using clinically relevant conditions. Methods Primary CLL B-cells and two CLL cell lines were exposed to a dose that is clinically achievable by AA oral administration (250 μM), and cell death and potential mechanisms were assessed. The role of the protective CLL microenvironment was studied. Synergistic interaction between AA and CLL approved drugs (Ibrutinib, Idelalisib and Venetoclax) was also evaluated. Results Ascorbic acid is cytotoxic for CLL B-cells at low dose (250 μM) but spares healthy B-cells. Ascorbic-acid-induced cytotoxicity involved pro-oxidant damage through the generation of reactive oxygen species in the extracellular media and in CLL cells, and induced caspase-dependent apoptosis. We also found that AA treatment overcame the supportive survival effect provided by microenvironment including bone marrow mesenchymal stem cells, T-cell cues (CD40L + IL-4), cytokines and hypoxia. Our data suggest that resistance to AA could be mediated by the expression of the enzyme catalase in some CLL samples and by the glucose metabolite pyruvate. We also demonstrated that AA synergistically potentiates the cytotoxicity of targeted therapies used in or being developed for CLL. Conclusion These preclinical results point to AA as an adjuvant therapy with potential to further improve CLL treatments in combination with targeted therapies.
Background: One of the major difficulties in cleft palate repair is the requirement for several surgical procedures and autologous bone grafting to form a bony bridge across the cleft defect. Engineered tissue, composed of a biomaterial scaffold and multipotent stem cells, may be a useful alternative for minimizing the non-negligible risk of donor site morbidity. The present study was designed to confirm the healing and osteogenic properties of a novel alginate-based hydrogel in palate repair. Methods: Matrix constructs, seeded with allogeneic bone marrow–derived mesenchymal stem cells (BM-MSCs) or not, were incorporated into a surgically created, critical-sized cleft palate defect in the rat. Control with no scaffold was also tested. Bone formation was assessed using microcomputed tomography at weeks 2, 4, 8, and 12 and a histologic analysis at week 12. Results: At 12 weeks, the proportion of bone filling associated with the use of hydrogel scaffold alone did not differ significantly from the values observed in the scaffold-free experiment (61.01% ± 5.288% versus 36.91% ± 5.132%; p = 0.1620). The addition of BM-MSCs stimulated bone formation not only at the margin of the defect but also in the center of the implant. Conclusions: In a relevant in vivo model of cleft palate in the rat, we confirmed the alginate-based hydrogel’s biocompatibility and real advantages for tissue healing. Addition of BM-MSCs stimulated bone formation in the center of the implant, demonstrating the new biomaterial’s potential for use as a bone substitute grafting material for cleft palate repair.
In bone tissue engineering, autologous cells are combined with osteoconductive scaffolds and implanted into bone defects. The major challenge is the lack of post-implantation vascular growth into biomaterial. The objective of the present study was to develop a new alginate-based hydrogel that enhances the regeneration of bone defects after surgery. The viability of human bone marrow-derived mesenchymal stem cells (BM-MSCs) or human endothelial cells (ECs) cultured alone or together on the hydrogel was analyzed for 24 and 96 h. After seeding, the cells self-assembled and aggregated to form clusters. For functional validation, empty or cellularized hydrogel matrices were implanted ectopically at subcutaneous sites in nude mice. After 2 months, the matrices were explanted. Transplanted human cells were present, and we observed vessels expressing human von Willebrand factor (resulting from the incorporation of transplanted ECs into neovessels and/or the differentiation of BM-MSCs into ECs). The addition of BM-MSCs improved host vascularization and neovessel formation from human cells, relative to ECs alone. Although we did not observe bone formation, the transplanted BM-MSCs were able to differentiate into osteoblasts. This new biomaterial provided an appropriate three-dimensional environment for transplanted cells and has a high angiogenic capacity and an osteogenic potential.
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