Luc Marchal

Interest of second harmonic generation imaging for diagnosis in thick and opaque tissue.

In articular hyaline cartilage, chondrocytes are surrounded by an extracellular matrix which is mainly composed by collagen and proteoglycanes. Pathological specimens show a partial or complete degradation of this matrix. Therefore, it could be interesting to know how mechanical or biochemical constraints applied to cartilage specimens induce modifications of the cartilage network. Multiphoton technology combined to Second Harmonic Generation (SHG) enables to image cartilage specimens in a non-invasive mode with high resolution at deep penetration. By placing a band pass filter in front of the transmitted light detector, SHG signal with frequency doubled can be isolated for a new contrast imaging. SHG (second harmonic generation) is a diffusion process generated from organized structures and does not need any fluorescent staining. Due to their non-centrosymetric structure, collagen fibrilles present a high second-order non-linear susceptibility and thus give rise to a strong SHG signal when exposed to high enough electric fields produced by a focal point of a femtosecond pulsed laser (multiphoton microscopy). As the extracellular matrix of cartilage is in part constituted by collagen fibers, it can be imaged with this contrast tool. The intensity of SHG signals strongly depends on the organization of collagen fibers. Thus a modification of the extracellular matrix in terms of 3D-organization of collagen induced by mechanical stress can be shown with this contrast tool.

Efficient generation of CD34+ progenitor-derived dendritic cells from G-CSF-mobilized peripheral mononuclear cells does not require hematopoietic stem cell enrichment

As a result of their potent antigen‐presentation function, dendritic cells (DC) are important tools for cell therapy programs. In vitro‐generated DC from enriched CD34+ hematopoietic stem cells (HSC; enriched CD34 DC) have already proven their efficiency in Phase I/II clinical trials. Here, we investigated whether enrichment of CD34+ HSC before the onset of culture was absolutely required for their differentiation into DC. With this aim, we developed a new two‐step culture method. PBMC harvested from G‐CSF‐mobilized, healthy patients were expanded for 7 days during the first step, with early acting cytokines, such as stem cell factor, fetal liver tyrosine kinase 3 ligand (Flt‐3L), and thrombopoietin. During the second step, expanded cells were then induced to differentiate into mature DC in the presence of GM‐CSF, Flt‐3L, and TNF‐α for 8 days, followed by LPS exposure for 2 additional days. Our results showed that the rate of CD34+/CD38+/lineageneg cells increased 19.5 ± 10‐fold (mean±sd) during the first step, and the expression of CD14, CD1a, CD86, CD80, and CD83 molecules was up‐regulated markedly following the second step. When compared with DC generated from enriched CD34+ cells, which were expanded for 7 days before differentiation, DC derived from nonenriched peripheral blood stem cells showed a similar phenotye but higher yields of production. Accordingly, the allogeneic stimulatory capacity of the two‐step‐cultured DC was as at least as efficient as that of enriched CD34 DC. In conclusion, we report herein a new two‐step culture method that leads to high yields of mature DC without any need of CD34+ HSC enrichment.

Modeling pH-zone refining countercurrent chromatography: A dynamic approach☆

A model based on mass transfer resistances and acid-base equilibriums at the liquid-liquid interface was developed for the pH-zone refining mode when it is used in countercurrent chromatography (CCC). The binary separation of catharanthine and vindoline, two alkaloids used as starting material for the semi-synthesis of chemotherapy drugs, was chosen for the model validation. Toluene/CH3CN/water (4/1/5, v/v/v) was selected as biphasic solvent system. First, hydrodynamics and mass transfer were studied by using chemical tracers. Trypan blue only present in the aqueous phase allowed the determination of the parameters τextra and Pe for hydrodynamic characterization whereas acetone, which partitioned between the two phases, allowed the determination of the transfer parameter k0a. It was shown that mass transfer was improved by increasing both flow rate and rotational speed, which is consistent with the observed mobile phase dispersion. Then, the different transfer parameters of the model (i.e. the local transfer coefficient for the different species involved in the process) were determined by fitting experimental concentration profiles. The model accurately predicted both equilibrium and dynamics factors (i.e. local mass transfer coefficients and acid-base equilibrium constant) variation with the CCC operating conditions (cell number, flow rate, rotational speed and thus stationary phase retention). The initial hypotheses (the acid-base reactions occurs instantaneously at the interface and the process is mainly governed by mass transfer) are thus validated. Finally, the model was used as a tool for catharanthine and vindoline separation prediction in the whole experimental domain that corresponded to a flow rate between 20 and 60 mL/min and rotational speeds from 900 and 2100 rotation per minutes.

Centrifugal partition extraction of β-carotene from Dunaliella salina for efficient and biocompatible recovery of metabolites.

A biocompatible extraction method for β-carotene recovery from the microalga Dunaliella salina was studied. The centrifugal partition extraction was used for liquid-liquid mass transfer intensification during continuous extraction. Different solvents and process parameters were compared. Ethyl oleate extraction with 5% dichloromethane achieved a 65% β-carotene recovery with the least amount of cell damage as more than 65% of the cells remained viable as demonstrated by photosynthesis activity measurements.

Influences of construct properties on the proliferation and matrix synthesis of dedifferentiated chondrocytes cultured in alginate gel

To investigate whether the chondrocytes-alginate construct properties, such as cell seeding density and alginate concentration might affect the redifferentiation, dedifferentiated rat articular chondrocytes were encapsulated at low density (LD: 3 x 10(6) cells/ml) or high density (HD: 10 x 10(6) cells/ml) in two different concentrations of alginate gel (1.2% or 2%, w/v) to induce redifferentiation. Cell viability and cell proliferation of LD culture was higher than those of HD culture. The increase in alginate gel concentration did not make an obvious difference in cell viability, but reduced cell proliferation rate accompanied with the decrease of cell population in S phase and G2/M phase. Scan electron microscopy observation revealed that chondrocytes maintained round in shape and several direct cell-cell contacts were noted in HD culture. In addition, more extracellular matrix was observed in the pericellular region of chondrocytes in 2% alginate culture than those in 1.2% alginate culture. The same tendency was found for the synthesis of collagen type II. No noticeable expression of collagen type I was detected in all constructs at the end of 28-day cultures. These results suggested that construct properties play an important role in the process of chondrocytes redifferentiation and should be considered for creating of an appropriate engineered articular cartilage.

Interest of multimodal imaging in tissue engineering

One of the major limitations when imaging biological tissues results in their thickness and opacity. To overcome these limitations and to visualize some structures of interest and to precise the relationship between the structure and the biological function [6], new imaging methods have been developed. Confocal Laser Scanning Microscopy (CLSM) is the reference technique for biological imaging. But CLSM induces some problems of photo toxicity for tissue which are observed in hard conditions. To overcome this gap, linear optical microscopy also called multiphoton microscopy continues to prove its useful for imaging live tissues in particular with reduced toxicity [9,10]. Multiphoton imaging is noninvasive, by keeping the cells alive and enables to be close to physiological conditions and allows a deeper penetration into samples [3,11] than more usual UV-visible techniques [7]. MP-excitation needs a quasi simultaneous absorption of two (or 3) photons, that means a spatio-temporal confinement of photons. A way to satisfy it and to bring a molecule into its excited state consists in using short impulsions at very high frequency. To come back to its fundamental state, many desexcitation ways among which fluorescence emission are possible. The emitted signal is specific to a molecule and its physicochemical environment and can be resolved as well spatially as temporally or spectrally. Some new tools have been developed to obtain complementary information. FLIM (Fluorescence Lifetime Imaging Microscopy) provides a discrimination of molecules on their different lifetimes (contrast image) whereas their emission spectra overlap. Importantly, the lifetime value measured for by FLIM is a property of individual fluorescent molecules and is therefore largely independent of fluorophore concentration and photobleaching [5]. In addition, the emission of specific fluorophores can be contrasted against a strong auto-fluorescence background as encountered in many biological tissues.

Interest of Second Harmonic Generation imaging to study collageneous matrix modification in osteoarthritis disease

Cartilage degenerative diseases like osteoarthritis affect the organization of the biological extracellular matrix (ECM) surrounding chondrocytes. This ECM is mainly composed by collagen giving rise to a strong Second Harmonic Generation (SHG) Signal, due to its high non linear susceptibility. Mechanical stress leads to perturbation of the collagen network comparable to modification occurring in disease. To be sure that SHG signal comes specifically from the collagen network, the enzymatical action of Collagenase was followed. We clearly noted the decrease of the collagen specific signal according to incubation time due to enzymatic degradation. To characterize structural modification on the arrangement of collagen fibers in the ECM, we used image analysis based on co-occurrence matrix (Haralick). Textural features give information like homogeneity (Angular Second Moment) or size of textural elements (Inverse Difference Moment, Correlation). Samples submitted to compression are characterized by higher Correlation, associated with a decrease of IDM and ASM. Those evolutions suggest the presence of long linear structures, an effect of packing of collagen fibrils and the apparition of nodes where the density of collagen is important versus areas showing a lack of molecules. Collagen I, II and VI are biomarkers characterising disease states since its presence is increased in pathological cartilage (osteoarthritis). Fluorescence Lifetime Imaging Microscopy (FLIM) associated to Spectral and SHG analysis confirmed the presence of Collagen I and II in the extracellular and Collagen VI in the pericellular matrix of chondrocytes. SHG, FLIM and Spectral Imaging combined with multiphoton excitation enable tissue imaging at deep penetration. We pointed out a local modification of the ECM of cartilage without any labelling (SHG) under mechanical stress. Thus the association of all these techniques represents a potential diagnosis tool for disorganization of collagen.