Zied Souguir

Hydrophobically modified xanthan: an amphiphilic but not associative polymer.

Hydrophobic octyl moieties have been grafted in various densities onto the carboxylic acid functions of xanthan under its ordered conformation. The outcoming amphiphilic and associative properties were studied by fluorescence spectroscopy and rheology. Results showed that the conformation of xanthan is not affected by the chemical modification and remains the same as the native one. Additionally, xanthan derivatives do not show any viscoelastic enhancement; nevertheless, their dynamics is strongly slowed down: the higher the grafting density, the slower the relaxation. We proved that hydrophobically modified xanthan, even being amphiphilic, does not exhibit any additional associating properties compared to the unmodified xanthan. The high stiffness of xanthan helices does not allow the derivatives to adopt the organization usually observed for flexible amphiphilic polymers. On the basis of these observations, a model depicting such a singular behavior is proposed.

Formation of Brown Lines in Paper : Characterization of Cellulose Degradation at the Wet-Dry Interface

Brown lines were generated at the wet-dry interface on Whatman paper No. 1 by suspending the sheet vertically in deionized water. Formic acid and acetic acid were quantified in three areas of the paper defined by the wet-dry boundary (above, below, and at the tideline) using capillary zone electrophoresis with indirect UV detection. Their concentration increased upon accelerated aging of the paper and was highest in the tideline. The hydroperoxides have been quantified using reverse phase high performance liquid chromatography with UV detection based on the determination of triphenylphosphine oxide produced from the reaction with triphenylphosphine, and their highest concentration was found in the tideline as well. For the first time, it was shown that various types of hydroperoxides were present, water-soluble and non-water-soluble, most probably in part hydroperoxide functionalized cellulose. After accelerated aging, a significant increase in hydroperoxide concentration was found in all the paper areas. The molar masses of cellulose determined using size-exclusion chromatography with multiangle light scattering detection showed that, upon aging, cellulose degraded significantly more in the tideline area than in the other areas of the paper. The area below the tideline was more degraded than the area above. A kinetic study of the degradation of cellulose allowed determining the constants for glycosidic bond breaking in each of the areas of the paper.

A biomimetic hydrogel functionalized with adipose ECM components as a microenvironment for the 3D culture of human and murine adipocytes

The lack of relevant in vitro models for adipose tissue makes necessary the development of a more physiological environment providing spatial and chemical cues for the effective maturation of adipocytes. We developed a biofunctionalized hydrogel with components of adipose extracellular matrix: collagen I, collagen VI, and the cell binding domain of fibronectin and we compared it to usual 2D cultures on plastic plates. This scaffold allowed 3D culture of mature adipocytes from the preadipocytes cell lines 3T3‐L1 and 3T3‐F442A, as well as primary Human White Preadipocytes (HWP), acquiring in vivo‐like organization, with spheroid shaped adipocytes forming multicellular aggregates. The size of these aggregates increased with time up to 120 μm in diameter after 4 weeks of maturation, with good viability. Significantly higher lipogenic activity (up to 20‐fold at day 28 for HWP cultures) and differentiation rates were also observed compared to 2D. Gene expression analyses highlighted earlier differentiation and complete maturation of 3D HWP compared to 2D, reinforced by the expression of Perilipin protein after 21 days of nutrition. This increase in adipocytes phenotypic and genotypic markers made this scaffold‐driven culture as a robust adipose 3D model. Retinoic acid inhibition of lipogenesis in HWP or isoprenalin and caffeine induction of lipolysis performed on mouse 3T3‐F442A cells, showed higher doses of molecules than typically used in 2D, underlying the physiologic relevance of this 3D culture system. Biotechnol. Bioeng. 2017;114: 1813–1824.

Evaluation by quantitative image analysis of anticancer drug activity on multicellular spheroids grown in 3D matrices

Pharmacological evaluation of anticancer drugs using 3D in vitro models provides invaluable information for predicting in vivo activity. Artificial matrices are currently available that scale up and increase the power of such 3D models. The aim of the present study was to propose an efficient and robust imaging and analysis pipeline to assess with quantitative parameters the efficacy of a particular cytotoxic drug. HCT116 colorectal adenocarcinoma tumor cell multispheres were grown in a 3D physiological hyaluronic acid matrix. 3D microscopy was performed with structured illumination, whereas image processing and feature extraction were performed with custom analysis tools. This procedure makes it possible to automatically detect spheres in a large volume of matrix in 96-well plates. It was used to evaluate drug efficacy in HCT116 spheres treated with different concentrations of topotecan, a DNA topoisomerase inhibitor. Following automatic detection and quantification, changes in cluster size distribution with a topotecan concentration-dependent increase of small clusters according to drug cytotoxicity were observed. Quantitative image analysis is thus an effective means to evaluate and quantify the cytotoxic and cytostatic activities of anticancer drugs on 3D multicellular models grown in a physiological matrix.

Abstract 4266: Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds

Extracellular matrix (ECM) characteristics, including stiffness, porosity, composition and spatial interaction with the surrounding cells and soluble factors are key components for cell growth in a tissue microenvironment. However, poor performance of 2D in vitro systems and animal models demands physiologically relevant well controlled 3D platforms for mechanistic assays, drug resistant phenotypes, new drug efficacy, toxicity assessment. Technical limitations in the current use of multicellular spheroids prevent their widespread use in cancer research and drug development. Existing systems for spheroid formation require lengthy processing times, and make simple tasks like media exchange, cell retrieval and microscopy analysis challenging. An ideal 3D cell culture system would form spheroids in an in vivo like microenvironment, while being easy to handle and compatible with all analytical methods. To address this unmet needs, we use a controlled hyaluronic acid-based scaffolds for spheroid formation. We have enriched 2 hyaluronan scaffolds with other components of the ECM such as collagen I, collagen IV, collagen VI, RGDs motif or galactosamine. Functionalised scaffolds overpass the 2D flat culture limitations by recreating cell/cell interactions and cell/matrix interaction to recreate a more physiologically authentic 3D architecture. The 2 functionalized scaffolds recapitulate the microenvironment for Hepatocyte and Adipocyte growth. Once formed, the spheroids can be cultured long-term, the scaffold is transparent allowing reproducible High Content Screening, the spheroids and the cells can be retrieve, avoiding the technical issues of other 3D systems to retain samples. Moreover, the scaffold is compatible with fluorescence/luminescent kit and immunofluorescent microscopy. We demonstrated this technology using cell lines, primary cells (adipocytes, hepatocytes). We assayed the spheroids over time using various endpoint spheroid morphology, growth and viability, resistance to anti-cancer drug, relevant cell organization formation and toxicity endpoint. Thus, this study introduces functionalized HA scaffold for the use of in vitro culture model as that represent native cell environments is ready to ready to use and compatible with HTS and all analytical methods for drug development and compound screening. Citation Format: Pauline Pannetier, Fiona Louis, Zied Souguir, Agathe Devaux, Didier Le Cerf, Jean-Pierre Vannier, Elise Demange, Guillaume Vidal. Reproducible spheroid formation using functionalized hyaluronan 3D scaffolds. [abstract]. In: Proceedings of the 107th Annual Meeting of the American Association for Cancer Research; 2016 Apr 16-20; New Orleans, LA. Philadelphia (PA): AACR; Cancer Res 2016;76(14 Suppl):Abstract nr 4266.