Sterilization of Exopolysaccharides Produced by Deep-Sea Bacteria: Impact on Their Stability and Degradation

Polysaccharides are highly heat-sensitive macromolecules, so high temperature treatments are greatly destructive and cause considerable damage, such as a great decrease in both viscosity and molecular weight of the polymer. The technical feasibility of the production of exopolysaccharides by deep-sea bacteria Vibrio diabolicus and Alteromonas infernus was previously demonstrated using a bioproduct manufacturing process. The objective of this study was to determine which sterilization method, other than heat sterilization, was the most appropriate for these marine exopolysaccharides and was in accordance with bioprocess engineering requirements. Chemical sterilization using low-temperature ethylene oxide and a mixture of ionized gases (plasmas) was compared to the sterilization methods using gamma and beta radiations. The changes to both the physical and chemical properties of the sterilized exopolysaccharides were analyzed. The use of ethylene oxide can be recommended for the sterilization of polysaccharides as a weak effect on both rheological and structural properties was observed. This low-temperature gas sterilizing process is very efficient, giving a good Sterility Assurance Level (SAL), and is also well suited to large-scale compound manufacturing in the pharmaceutical industry

Development of thermo-responsive supports for the culture of adherent cells and their protease-free detachment

Les cellules adhérentes, dont les cellules souches, sont très prometteuses pour de nombreuses applications dans le domaine des biotechnologies. Toutefois, leur culture représente un défi puisque les méthodes enzymatiques et mécaniques, couramment utilisées pour l’étape de détachement du support de culture, présentent des inconvénients majeurs. Le but de ce travail de thèse a été de développer un support pour la culture de cellules adhérentes permettant leur détachement par modification de la température. Pour cela, nous avons produit des échantillons thermo-associatifs de xyloglucane, un polysaccharide naturel, par réaction avec une enzyme, la ß-galactosidase. Les échantillons obtenus ont été caractérisés par rhéologie, viscosimètrie, diffusion de la lumière et chromatographie par perméation de gel. Nous nous sommes aussi intéressés à l’évaluation de méthodes de stérilisation efficaces capables de préserver les propriétés du polymère. De plus, afin de rendre ce polymère bioadhésif, nous avons étudié son activation et sa fonctionnalisation par un peptide contenant le motif arginine-glycine-aspartate (RGD). Nous avons montré que des cellules modèles A375 surexprimant le récepteur pour le RGD sont capables d’adhérer et de proliférer à 37°C sur des films formés à partir de xyloglucane modifié par du RGD. Leur détachement est possible à température ambiante. L'utilisation de ce nouveau support pour la culture de cellules souches embryonnaires humaines, la production d’un système pour la libération contrôlée de facteurs de croissance ainsi que la conception de billes de taille micrométrique à base de xyloglucane font partie des perspectives de cette thèse.Adherent cells, including stem cells, hold great promise for many applications in biotechnology. However, cell detachment from standard culture surfaces remains a challenging task since both commonly used enzymatic and mechanical methods have major drawbacks. The aim of my PhD work was to develop a thermoresponsive culture surface supporting cell attachment and survival while enabling a temperature-assisted cell detachment. In order to achieve this goal, thermoresponsive xyloglucan was initially produced by partial galactose removal after reaction with the β-galactosidase enzyme. Samples were characterized by rheology, viscosimetry, light scattering analysis and size-exclusion chromatography. We were also interested in evaluating sterilization methods and their effect on the properties of the polymer. Additionally, in order to obtain a bioadhesive polymer from partially degalactosylated xyloglucan, we studied its functionalization with a peptide containing the arginine-glycine-aspartate (RGD) groupment motif. We have shown that A375 model cells overexpressing the RGD receptor were able to adhere and proliferate at 37 °C on xyloglucan-derived films grafted with RGD and their detachment at room temperature was also demonstrated. The culture of human embryonic stem cells, as well as the introduction of a controlled release system for growth factors and the development of thermoresponsive beads for cell culture comprise the perspectives of this thesis

Développement de nouveaux supports pour la culture de cellules adhérentes permettant leur détachement sans utilisation de protéases

Résumé soumis à confidentialitéAbstract not availableEVRY-Bib. électronique (912289901) / SudocSudocFranceF

Mechanism of Associations of Neutral Semiflexible Biopolymers in Water: The Xyloglucan Case Reveals Inherent Links

International audienceThe mechanisms of association between neutral xyloglucan chains extracted from tamarind seeds are explored. Mesoscale structures involving a few chains are evidenced and monitored by static light scattering and low-shear viscosity experiments as a function of the xyloglucan concentration, obtained from increasing the dilution of an initial dispersion. The mechanism of association is addressed by means of multiangle dynamic light scattering. The associations exist whether the chains are in the dilute regime or in the semi-dilute regime and are characteristic of weak interactions. Their progressive loosening by dilution is evidenced and their level depends only on the xyloglucan concentration. The associations are due to a mechanism inherent to the nature of the chain