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The main objective of this research was to define a structured and functionalized support for future biomedical applications (model of “low-density bioarray”). The experiments were carried out by using stereolithography process with a special SU-8 photoresist and the reproducibility of the method was studied by analyzing the surface profile of the support. Finally, a matrix of regular controlled sized wells was fabricated. Chemical reactions leading to covalent grafting were run to demonstrate that the inner surface of the wells remains still reactive after polymerization. The grafting of fluorophores with carboxylic functions activated by N-hydroxysuccinimide was studied as function of time, in order to determine the best reactions, conditions. Then, the grafting of two distinct fluorescent probes was led simultaneously inside the wells, showing the possibility of spatial localization of diverse reactions on the same support. The covalent and localized bindings were confirmed by fluorescence spectroscopy and microscopy analyses.

The photocatalytic decolorization of industrial textile dyes has been studied. The treatment was carried out on a solar reactor consisting in a flat active plane, tilted so as to face the sun and to allow the trickling of the water to be treated. Alternatively the reactor could be irradiated by an artificial source. After checking the system using salicylic acid, a conventional model molecule, the photocatalytic decolorization of Orange II, Yellow Drimarene, and Black Drimarene dyes was investigated. Artificial and solar irradiation gave comparable results although the heating by the sun reduced the amount of adsorption. The kinetics agrees with the Langmuir-Hinshelwood model and a discrepancy between adsorption constants deduced from the kinetic and adsorption experiments was interpreted by considering various types of adsorption sites. Orange II and Drimarene dyes decolorization kinetics are opposite limiting cases of the above model, as being of order 0 and 1 with respect to the dye, respectively.

Here, we describe a protocol for producing micropatterned porous membranes which can be used for combinatorial cell-based assays. We use contact printing to pattern the surface of a porous filter membrane with a thin layer of polydimethylsiloxane (PDMS). This allows the porosity of the filter membrane to be altered at selected locations. Cells can be grown on one side of the filter membrane, while drugs and reagents can be deposited on the porous areas of the other side of the membrane. The reagents can diffuse through the pores of the membrane to the cells. The first part of the protocol describes how to design a stamp and use it to contact print PDMS. The second part describes how to create microprinted membranes for cell-based assays. The method is simple, highly customizable, can be performed at the bench, and can be used to perform combinatorial or time-dependent cell-based assays., (Copyright © 2014 Elsevier Inc. All rights reserved.)

In the search for new therapeutic chemicals, lab-on-a-chip systems have recently emerged as innovative and efficient tools for cell-based assays and high throughput screening. Here, we describe a novel, versatile and simple device for cell-based assays at the bench-top. We created spatial variations of porosity on the surface of a membrane filter by microcontact printing with a biocompatible polymer (PDMS). We called such systems Micro-Printed Membranes (μPM). Active compounds dispensed on the porous areas, where the membrane pores are not clogged by the polymer, can cross the membrane and reach cells growing on the opposite side. Only cells immediately below those porous areas could be stimulated by chemicals. We performed proof-of-principle experiments using Hoechst nuclear staining, calcein-AM cell viability assay and destabilization of the cytoskeleton organisation by cytochalasin B. Resulting fluorescent staining properly matched the drops positioning and no cross-contaminations were observed between adjacent tests. This well-less cell-based screening system is highly flexible by design and it enables multiple compounds to be tested on the same cell tissue. Only low sample volumes in the microlitre range are required. Moreover, chemicals can be delivered sequentially and removed at any time while cells can be monitored in real time. This allows the design of complex, sequential and combinatorial drug assays. μPMs appear as ideal systems for cell-based assays. We anticipate that this lab-on-chip device will be adapted for both manual and automated high content screening experiments.

In this chapter, from the engineering point of view, we introduce the results from our group and related research on three typical configurations of engineered liver tissues; cell sheet-based tissues, sheet-like macroporous scaffold-based tissues, and tissues based on special scaffolds that comprise a flow channel network. The former two do not necessitate in vitro prevascularization and are thus promising in actual human clinical trials for liver diseases that can be recovered by relatively smaller tissue mass. The third approach can implant a much larger mass but is still not yet feasible. In all cases, oxygen supply is the key engineering factor. For the first configuration, direct oxygen supply using an oxygen-permeable polydimethylsiloxane membrane enables various liver cells to exhibit distinct behaviors, complete double layers of mature hepatocytes and fibroblasts, spontaneous thick tissue formation of hepatocarcinoma cells and fetal hepatocytes. Actual oxygen concentration at the cell level can be strictly controlled in this culture system. Using this property, we found that initially low then subsequently high oxygen concentrations were favorable to growth and maturation of fetal cells. For the second configuration, combination of poly-L: -lactic acid 3D scaffolds and appropriate growth factor cocktails provides a suitable microenvironment for the maturation of cells in vitro but the cell growth is limited to a certain distance from the inner surfaces of the macropores. However, implantation to the mesentery leaves of animals allows the cells again to proliferate and pack the remaining spaces of the macroporous structure, suggesting the high feasibility of 3D culture of hepatocyte progenitors for liver tissue-based therapies. For the third configuration, we proposed a design criterion concerning the dimensions of flow channels based on oxygen diffusion and consumption around the channel. Due to the current limitation in the resolution of 3D microfabrication processes, final cell densities were less than one-tenth of those of in vivo liver tissues; cells preferentially grew along the surfaces of the channels and this fact suggested the necessity of improved 3D fabrication technologies with higher resolution. In any case, suitable oxygen supply, meeting the cellular demand at physiological concentrations, was the most important factor that should be considered in engineering liver tissues. This enables cells to utilize aerobic respiration that produces almost 20 times more ATP from the same glucose consumption than anaerobic respiration (glycolysis). This also allows the cells to exhibit their maximum reorganization capability that cannot be observed in conventional anaerobic conditions.

To engineer reliable in vitro liver tissue equivalents expressing differentiated hepatic functions at a high level and over a long period of time, it appears necessary to have liver cells organized into a three-dimensional (3D) multicellular structure closely resembling in vivo liver cytoarchitecture and promoting both homotypic and heterotypic cell-cell contacts. In addition, such high density 3D hepatocyte cultures should be adequately supplied with nutrients and particularly with oxygen since it is one of the most limiting nutrients in hepatocyte cultures. Here we propose a novel but simple hepatocyte culture system in a microplate-based format, enabling high density hepatocyte culture as a stable 3D-multilayer. Multilayered co-cultures of hepatocytes and 3T3 fibroblasts were engineered on collagen-conjugated thin polydimethylsiloxane (PDMS) membranes which were assembled on bottomless frames to enable oxygen diffusion through the membrane. To achieve high density multilayered co-cultures, primary rat hepatocytes were seeded in large excess what was rendered possible due to the removal of oxygen shortage generally encountered in microplate-based hepatocyte cultures. Hepatocyte/3T3 fibroblasts multilayered co-cultures were maintained for at least 1 week; the so-cultured cells were normoxic and sustained differentiated metabolic functions like albumin and urea synthesis at higher levels than hepatocytes monocultures. Such a microplate-based cell culture system appears suitable for engineering in vitro miniature liver tissues for implantation, bioartificial liver (BAL) development, or chemical/drug screening., (Copyright © 2011 American Institute of Chemical Engineers (AIChE).)

Here we report the use of natural, chemically-unmodified, microfibrillated cellulose (MFC) as a matrix for hepatocyte culture. We developed an original cell-culture design composed of a thin 3D-microstructured fibrous substrate consisting of a MFC sheet coating a highly O(2)-permeable polydimethylsiloxane (PDMS) membrane. The MFC-coated PDMS membranes were obtained according to a simple process where cellulose fibres were deposited from an aqueous suspension on the PDMS surfaces and the films were dried under mild conditions. To enable oxygen diffusion through the membranes, they were assembled on bottomless frames ('O(2)+' condition). Rat hepatocytes primary-cultured on such MFC-PDMS membranes quickly organized themselves into large hemispherical 3D aggregates which were tightly anchored to the MFC sheets. In contrast, hepatocytes cultured on smooth PDMS membranes in the O(2)+ system (O(2)+, PDMS) organized into unstable 2D monolayers which easily detached from the surfaces. Hepatocyte 3D cultures obtained on MFC-PDMS membranes exhibited higher liver-specific functions over a 2-week culture period, as assessed by both the higher albumin secretion and urea synthesis rate. The MFC-PDMS membranes appear suitable for obtaining stably-attached and functional hepatocyte 3D cultures and appear interesting for drug/chemical screenings in a microplate format, but also for microfluidic applications.

In order to enhance the viability and the differentiated functions of primary hepatocytes in cultures, it appears important to have them organized within a three-dimensional (3D) structure which promotes extensive cell-cell contacts, but also to be adequately supplied with oxygen. Here, we report a simple methodology satisfying these two fundamental but sometimes conflicting issues: primary rat hepatocytes were cultured on polydimethylsiloxane (PDMS) membranes with 3D-pillared microstructures with various dimensions, so that the cells could organize themselves around the pillars into various kinds of 3D multicellular aggregates, while being continuously supplied with oxygen by diffusion through the PDMS membrane. As expected, under such conditions, hepatocyte cultures exhibited higher albumin secretion and urea synthesis rates. It appeared then that as the spacing decreased between the pillars, the cells were more stably organized into smaller spherical aggregates and displayed the highest albumin secretion rates. Such a simple design is likely to be included in a new drug/chemical screening system in a practical microplate format, but also appears applicable to microfluidic devices.

When considering high-density liver cell cultures, adequate delivery of oxygen to the cells appears particularly crucial since it is one of the most limiting parameters of hepatocytes' functions. Here we report on the effects of direct oxygenation through a gas-permeable polydimethylsiloxane membrane on liver-derived cell culture. We used highly proliferative human hepatoma Hep G2 cells to assess the growth-related limitation of such direct oxygen supply. As a consequence of the greater oxygen availability, the proliferation of Hep G2 cells was markedly enhanced, leading to the formation of a thick three-dimensional cellular multilayer. This was supported by the oxygen concentration profiles in the vicinity of the cell layers, predicted by numerical simulations. The applied cells also displayed an increased functionality as assessed by the high-albumin production rates, with per well-based albumin secretion reaching after 15 days 50 times that of the cells cultured in conventional polystyrene plates. Anaerobic glycolysis was in the same time significantly reduced as assessed by both the reduced glucose consumption and lactate production rates. Direct oxygenation through such a gas-permeable membrane appears suitable for engineering functional thick liver tissues for in vivo implantation, as well as for microplate-based chemical/drug screening applications.

Vitamin A is used as a generic term for all vitamin A derivatives with retinol-like biological activity. Retinol is the main parent compound for vitamin A. It derives from carotenoids (provitamin A) and also directly from the pre-formed vitamin A contained in the diet. The term "retinoid" is a generic descriptor of compounds structurally related to vitamin A and the synthetic analogues of retinol with or without biological activity. Retinoic acid is the active cellular catabolite. Vitamin A/retinoids have been given cancer-preventive functions and subsequently used in clinical trials to reduce lung cancer incidence in high-risk individuals. The results obtained have been in contradiction with both in vivo and in vitro promising studies. It seems therefore necessary to develop a better understanding of the vitamin A/retinoids signalling pathways in the lung. With this aim, we summarise the relevant knowledge focussed on the lung.