Three-dimensional in vitro culture models in oncology research.

Cancer is a multifactorial disease that is responsible for 10 million deaths per year. The intra- and inter-heterogeneity of malignant tumors make it difficult to develop single targeted approaches. Similarly, their diversity requires various models to investigate the mechanisms involved in cancer initiation, progression, drug resistance and recurrence. Of the in vitro cell-based models, monolayer adherent (also known as 2D culture) cell cultures have been used for the longest time. However, it appears that they are often less appropriate than the three-dimensional (3D) cell culture approach for mimicking the biological behavior of tumor cells, in particular the mechanisms leading to therapeutic escape and drug resistance. Multicellular tumor spheroids are widely used to study cancers in 3D, and can be generated by a multiplicity of techniques, such as liquid-based and scaffold-based 3D cultures, microfluidics and bioprinting. Organoids are more complex 3D models than multicellular tumor spheroids because they are generated from stem cells isolated from patients and are considered as powerful tools to reproduce the disease development in vitro. The present review provides an overview of the various 3D culture models that have been set up to study cancer development and drug response. The advantages of 3D models compared to 2D cell cultures, the limitations, and the fields of application of these models and their techniques of production are also discussed.

In vitro three-dimensional cell cultures for bone sarcomas.

Bone sarcomas are rare tumour entities that arise from the mesenchyme most of which are highly heterogeneous at the cellular, genetic and epigenetic levels. The three main types are osteosarcoma, Ewing sarcoma, and chondrosarcoma. These oncological entities are characterised by high morbidity and mortality and an absence of significant therapeutic improvement in the last four decades. In the field of oncology, in vitro cultures of cancer cells have been extensively used for drug screening unfortunately with limited success. Indeed, despite the massive knowledge acquired from conventional 2D culture methods, scientific community has been challenged by the loss of efficacy of drugs when moved to clinical trials. The recent explosion of new 3D culture methods is paving the way to more relevant in vitro models mimicking the in vivo tumour environment (e.g. bone structure) with biological responses close to the in vivo context. The present review gives a brief overview of the latest advances of the 3D culture methods used for studying primary bone sarcomas.

An easy-to-build and re-usable microfluidic system for live-cell imaging.

BACKGROUND: Real-time monitoring of cellular responses to dynamic changes in their environment or to specific treatments has become central to cell biology. However, when coupled to live-cell imaging, such strategies are difficult to implement with precision and high time resolution, and the simultaneous alteration of multiple parameters is a major challenge. Recently, microfluidics has provided powerful solutions for such analyses, bringing an unprecedented level of control over the conditions and the medium in which cells under microscopic observation are grown. However, such technologies have remained under-exploited, largely as a result of the complexity associated with microfabrication procedures. RESULTS: In this study, we have developed simple but powerful microfluidic devices dedicated to live-cell imaging. These microsystems take advantage of a robust elastomer that is readily available to researchers and that presents excellent bonding properties, in particular to microscopy-grade glass coverslips. Importantly, the chips are easy-to-build without sophisticated equipment, and they are compatible with the integration of complex, customized fluidic networks as well as with the multiplexing of independent assays on a single device. We show that the chips are re-usable, a significant advantage for the popularization of microfluidics in cell biology. Moreover, we demonstrate that they allow for the dynamic, accurate and simultaneous control of multiple parameters of the cellular environment. CONCLUSIONS: While they do not possess all the features of the microdevices that are built using complex and costly procedures, the simplicity and versatility of the chips that we have developed make them an attractive alternative for a range of applications. The emergence of such devices, which can be fabricated and used by any laboratory, will provide the possibility for a larger number of research teams to take full advantage of these new methods for investigating cell biology.

A drug-compatible and temperature-controlled microfluidic device for live-cell imaging.

Monitoring cellular responses to changes in growth conditions and perturbation of targeted pathways is integral to the investigation of biological processes. However, manipulating cells and their environment during live-cell-imaging experiments still represents a major challenge. While the coupling of microfluidics with microscopy has emerged as a powerful solution to this problem, this approach remains severely underexploited. Indeed, most microdevices rely on the polymer polydimethylsiloxane (PDMS), which strongly absorbs a variety of molecules commonly used in cell biology. This effect of the microsystems on the cellular environment hampers our capacity to accurately modulate the composition of the medium and the concentration of specific compounds within the microchips, with implications for the reliability of these experiments. To overcome this critical issue, we developed new PDMS-free microdevices dedicated to live-cell imaging that show no interference with small molecules. They also integrate a module for maintaining precise sample temperature both above and below ambient as well as for rapid temperature shifts. Importantly, changes in medium composition and temperature can be efficiently achieved within the chips while recording cell behaviour by microscopy. Compatible with different model systems, our platforms provide a versatile solution for the dynamic regulation of the cellular environment during live-cell imaging.

Investigation of expression and activity levels of primary rat hepatocyte detoxication genes under various flow rates and cell densities in microfluidic biochips.

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.

Quantitative full-colour transmitted light microscopy and dyes for concentration mapping and measurement of diffusion coefficients in microfluidic architectures.

A simple and versatile methodology has been developed for the simultaneous measurement of multiple concentration profiles of colourants in transparent microfluidic systems, using a conventional transmitted light microscope, a digital colour (RGB) camera and numerical image processing combined with multicomponent analysis. Rigorous application of the Beer-Lambert law would require monochromatic probe conditions, but in spite of the broad spectral bandwidths of the three colour channels of the camera, a linear relation between the measured optical density and dye concentration is established under certain conditions. An optimised collection of dye solutions for the quantitative optical microscopic characterisation of microfluidic devices is proposed. Using the methodology for optical concentration measurement we then implement and validate a simplified and robust method for the microfluidic measurement of diffusion coefficients using an H-filter architecture. It consists of measuring the ratio of the concentrations of the two output channels of the H-filter. It enables facile determination of the diffusion coefficient, even for non-fluorescent molecules and nanoparticles, and is compatible with non-optical detection of the analyte.

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

We 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.

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 xenobiotic's 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 CYP1A1/2's 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 xenobiotic's 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.

On-chip multi-electrochemical sensor array platform for simultaneous screening of nitric oxide and peroxynitrite.

In this work we report on the design, microfabrication and analytical performances of a new electrochemical sensor array (ESA) which allows for the first time the simultaneous amperometric detection of nitric oxide (NO) and peroxynitrite (ONOO(-)), two biologically relevant molecules. The on-chip device includes individually addressable sets of gold ultramicroelectrodes (UMEs) of 50 µm diameter, Ag/AgCl reference electrode and gold counter electrode. The electrodes are separated into two groups; each has one reference electrode, one counter electrode and 110 UMEs specifically tailored to detect a specific analyte. The ESA is incorporated on a custom interface with a cell culture well and spring contact pins that can be easily interconnected to an external multichannel potentiostat. Each UME of the network dedicated to the detection of NO is electrochemically modified by electrodepositing thin layers of poly(eugenol) and poly(phenol). The detection of NO is performed amperometrically at 0.8 V vs. Ag/AgCl in phosphate buffer solution (PBS, pH = 7.4) and other buffers adapted to biological cell culture, using a NO-donor. The network of UMEs dedicated to the detection of ONOO(-) is used without further chemical modification of the surface and the uncoated gold electrodes operate at -0.1 V vs. Ag/AgCl to detect the reduction of ONOOH in PBS. The selectivity issue of both sensors against major biologically relevant interfering analytes is examined. Simultaneous detection of NO and ONOO(-) in PBS is also achieved.

In situ micropatterning technique by cell crushing for co-cultures inside microfluidic biochips.

To perform dynamic cell co-culture on micropatterned areas, we have developed a new type of "on chip and in situ" micropatterning technique. The microchip is composed of a 200 microm thick PDMS (polydimethylsiloxane) chamber at the top of which are located 100 mum thick microstamps. The PDMS chamber is bonded to a glass slide. After sterilization and cell adhesion processes, a controlled force is applied on the top of the PDMS chamber. Mechanically, the microstamps come into contact of the cells. Due to the applied force, the cells located under the microstamps are crushed. Then, a microfluidic perfusion is applied to rinse the microchip and remove the detached cells. To demonstrate the potential of this technique, it was applied successfully to mouse fibroblasts (Swiss 3T3) and liver hepatocarcinoma (HepG2/C3a) cell lines. Micropatterned areas were arrays of octagons of 150, 300 and 500 microm mean diameter. The force was applied during 30 to 60s depending on the cell types. After cell crushing, when perfusion was applied, the cells could successfully grow over the patterned areas. Cultures were successfully performed during 72 h of perfusion. In addition, monolayers of HepG2/C3a were micropatterned and then co cultured with mouse fibroblasts. Numerical simulations have demonstrated that the presence of the microstamps at the top of the PDMS chamber create non uniform flow and shear stress applied on the cells. Once fabricated, the main advantage of this technique is the possibility to use the same microchip several times for cell micropatterning and microfluidic co-cultures. This protocol avoids complex and numerous microfabrication steps that are usually required for micropatterning and microfluidic cell culture in the same time.

Development of a renal microchip for in vitro distal tubule models.

Current developments in tissue engineering and microtechnology fields have allowed the proposal of pertinent tools, microchips, to investigate in vitro toxicity. In the framework of the proposed REACH European directive and the 3R recommendations, the purpose of these microtools is to mimic organs in vitro to refine in vitro culture models and to ultimately reduce animal testing. The microchip consists of functional living cell microchambers interconnected by a microfluidic network that allows continuous cell feeding and waste removal controls by fluid microflow. To validate this approach, Madin Darby Canine Kidney (MDCK) cells were cultivated inside a polydimethylsiloxane microchip. To assess the cell proliferation and feeding, the number of inoculated cells varied from 5 to 10 x 10(5) cells/microchip (corresponding roughly to 2.5 to 5 x 10(5) cells/cm2) and from four flow rates 0, 10, 25, and 50 microL/min were tested. Morphological observations have shown successful cell attachment and proliferation inside the microchips. The best flow rate appears to be 10 microL/min with which the cell population was multiplied by about 2.2 +/- 0.1 after 4 days of culture, including 3 days of perfusion (in comparison to 1.7 +/- 0.2 at 25 microL/min). At 10 microL/min flow rate, maximal cell population reached about 2.1 +/- 0.2 x 10(6) (corresponding to 7 +/- 0.7 x 10(7) cells/cm(3)). The viability, assessed by trypan blue and lactate deshydrogenase measurements, was found to be above 90% in all experiments. At 10 microL/min, glucose monitoring indicated a cell consumption of 16 +/- 2 microg/h/10(6) cells, whereas the glutamine metabolism was demonstrated with the production of NH3 by the cells about 0.8 +/- 0.4 micromol/day/10(6) cells. Augmentation of the flow rate appeared to increase the glucose consumption and the NH3 production by about 1.5- to 2-fold, in agreement with the tendencies reported in the literature. As a basic chronic toxicity assessment in the microchips, 5 mM and 10 mM ammonium chloride loadings, supplemented in the culture media, at 0, 10, and 25 micaroL/min flow rates were performed. At 10 microL/min, a reduction of 35% of the growth ratio with 5 mM and of 50% at 10 mM was found, whereas at 25 microL/min, a reduction of 10% with 5 mM and of 30% at 10 mM was obtained. Ammonium chloride contributed to increase the glucose consumption and to reduce the NH3 production. The microchip advantages, high surface/volume ratio, and dynamic loadings, coupled with the concordance between the present and literature results dealing with ammonia/ammonium effects on MDCK illustrate the potential of our microchip for wider in vitro chronic toxicity investigations.

Selective control of liver and kidney cells migration during organotypic cocultures inside fibronectin-coated rectangular silicone microchannels.

In this work, the behaviors of embryonic liver and kidney explants were studied inside rectangular polydimethylsiloxane (PDMS) microchannels. The organs were cultured under monoculture and coculture conditions on PDMS coated with or without fibronectin. The results demonstrated that the migration of cells from both organs is dependent on culture conditions and thus can be selectively controlled. In liver monocultures without fibronectin, cell migration in the microchannels resulted in the formation of a dense 3D tissue. Fibronectin reduced liver cell migration and enhanced the emergence of cells demonstrating typical hepatocyte phenotypes at the vicinity of the explant. The migration rate in liver-liver cocultures, with and without fibronectin, was roughly twice the rate of cells under monoculture conditions. In cocultures, both livers merged to form a large tissue in which the two initial organs could not be identified. In kidney monocultures, with and without fibronectin, we did not observe any migration inside the microchannels. Contrary to liver cells, kidney cell migration was triggered when both fibronectin coating and coculture with liver or another kidney explant were used. The migration was more largely observed in coculture with liver when compared to kidney-kidney cocultures. In the case of liver-kidney coculture with fibronectin, the progression of the kidney cells inside the microchannels appears as a displacement of the entire kidney explant in the direction of the liver. The liver cells did not move in those cases. After contact, we observed a complete merging of both liver and kidney explants. In contrast, for liver-kidney cocultures without fibronectin, only the liver moved toward the kidney.

Trends in the development of microfluidic cell biochips for in vitro hepatotoxicity.

Current developments in the technological fields of liver tissue engineering, bioengineering, biomechanics, microfabrication and microfluidics have lead to highly complex and pertinent new tools called "cell biochips" for in vitro toxicology. The purpose of "cell biochips" is to mimic organ tissues in vitro in order to partially reduce the amount of in vivo testing. These "cell biochips" consist of microchambers containing engineered tissue and living cell cultures interconnected by a microfluidic network, which allows the control of microfluidic flows for dynamic cultures, by continuous feeding of nutrients to cultured cells and waste removal. Cell biochips also allow the control of physiological contact times of diluted molecules with the tissues and cells, for rapid testing of sample preparations or specific addressing. Cell biochips can be situated between in vitro and in vivo testing. These types of systems can enhance functionality of cells by mimicking the tissue architecture complexities when compared to in vitro analysis but at the same time present a more rapid and simple process when compared to in vivo testing procedures. In this paper, we first introduce the concepts of microfluidic and biochip systems based on recent progress in microfabrication techniques used to mimic liver tissue in vitro. This includes progress and understanding in biomaterials science (cell culture substrate), biomechanics (dynamic cultures conditions) and biology (tissue engineering). The development of new "cell biochips" for chronic toxicology analysis of engineered tissues can be achieved through the combination of these research domains. Combining these advanced research domains, we then present "cell biochips" that allow liver chronic toxicity analysis in vitro on engineered tissues. An extension of the "cell biochip" idea has also allowed "organ interactions on chip", which can be considered as a first step towards the replacement of animal testing using a combined liver/lung organ model.

Guidance of liver and kidney organotypic cultures inside rectangular silicone microchannels.

We have studied the effect of rectangular polydimethylsiloxane (PDMS) microchannels on the behavior of embryonic liver and kidney explants maintained in contact with these microchannels. The microchannel widths were varied from 35 to 300 microm and depth from 45 to 135 microm. The growth of these tissue types were compared to the development on flat silicone and plastic control material. At seeding, due to the viscoelastic properties of both organs, "capillary-like filling" was observed inside the narrowest microchannels. In those cases, the tissues grew to a confluent layer joining the microchannels with no cell migration and proliferation inside the microchannels. In the largest microchannels, only a weak migration was observed and the cellular behavior appears quite similar to that of PDMS flat culture conditions. In intermediate geometries, we observed different tissue growth progressed inside those microchannels with an average growth properties inside the microchannels when compared to other sizes. The liver tissues velocity of up to 72 microm/day resulting to form a dense three-dimensional multicellular 'liver-like tissue'. Scanning electron microscopy (SEM) observations demonstrated that the tissue was organized like an epithelial layer with round cells embedded in an extracellular matrix. Liver cell mobility may result primarily from the activity of the marginal cells, whereas the sub-marginal cells appeared passively dragged. Parenchymal organization demonstrating differentiated states was also observed. Kidney grew mainly on the microchannel walls and the tissues never appeared dense and organized as the liver ones.

Constraining the connectivity of neuronal networks cultured on microelectrode arrays with microfluidic techniques: a step towards neuron-based functional chips.

In vitro culture of small neuronal networks with pre-defined topological features is particularly desirable when the electrical activity of such assemblies can be monitored for long periods of time. Indeed, it is hoped that such networks, with pre-determined connectivity, will provide unique insights into the structure/function relationship of biological neural networks and their properties of self-organization. However, the experimental techniques that have been developed so far for that purpose have either failed to provide very long-term pattern definition and retention, or they have not shown potential for integration into more complex microfluidic devices. To address this problem, three-dimensional microfluidic systems in poly(dimethylsiloxane) (PDMS) were fabricated and used in conjunction with both custom-made and commercially available planar microelectrode arrays (pMEAs). Various types of primary neuronal cell cultures were established inside these systems. Extracellular electrical signals were successfully recorded from all types of cells placed inside the patterns, and this bioelectrical activity was present for several weeks. The advantage of this approach is that it can be further integrated with microfluidic devices and pMEAs to yield, for example, complex neuron-based biosensors or chips for pharmacological screening.