Rationally designed polypeptide-based materials have advantages over other biomaterials for their ability to be engineered from the bottom-up, and to be tailored to the needs of specific potential applications. Based on previous works in the Woolfson laboratory on self- assembling peptide fibres (SAF), this research project focused on developing coiled-coil- based fibrillar hydrogel networks called hydrogelating SAFs (hSAFs), and applying them to more-complex biological systems, notably in cell culture. The hSAFs were tested as a three- dimensional cell-culture scaffold with the aim of mimicking the microenvironment provided by native extracellular matrix (ECM). hSAFs were shown to support growth and differentiation of neuronal cells. Furthermore, a design strategy was explored on how to couple short biomimetic peptides to these proteinogenic scaffolds to enhance cell-matrix interactions. This was achieved by integrating the well-studied cell-adhesion motif RGDS (Arg-Gly-Asp- Ser) to hSAFs using bioorthogonal copper-based click chemistry. Addition of such biomimetic peptides did not alter folding, fibre formation, and higher-order assembly (gelation) of the hSAFs. The resulting functionalised gels promoted early differentiation and longer neurite-like outgrowth in PC12 cells, and of primary rat hippocampal cells. The attached biomimetic peptide was functional and elicited the desired cellular response. Another short biomimetic peptides, i.e. the laminin-derived IKVAV (lIe-Lys-Val-Ala-Val), which supports neurite growth, was also successfully incorporated into hSAFs and gels. Thus, we have demonstrated a chemically defined, bottom-up approach to biological scaffold synthesis and assembly, and further the use of straightforward techniques to incorporate specific biomolecular signals under physiological conditions. Although conditions will need to be optimised for different cell types, hSAF gel formation and functionalisation via click chemistry promise a robust platform in cell-culture studies and potentially for tissue engineering.

The long term monitoring of cell cultures, to study their dynamic nature is important, although devices capable of doing this aren't available. Primiarily the reason for this is the bio-compatibility of the devices and this refers to both the devices' compatibility with the culture as well as its compatibility with the device. Glucose sensors fabricated in electropolymerised poly(phenol) were used for the study. The reproducibility of these devices was checked to assess their viability for large scale deployment. It was found that the intra-batch variation was within the norms of experimental error, although the inter-batch variation was significant. This is a feature that is inherent to hand made devices and some methods to mitigate this are discussed. The surface characterisation of the sensors showed that the polymer and enzyme sit mutually exclusive to each other on the surface. Using cyclic voltammetry, with ferrocyanideas the probe molecule, it was confirmed that the diffusion profile to the surface of the sensor was semi-infinite planar and that the electrochemistry was quasi-reversible. Infra-red and Raman spectroscopy and scanning electrochemical microscopy were used as well to study the films' morphology. The sensors were not toxic to 3T3 cell cultures, as the yield and cell viability were consistent with those of the control cultures. It was also seen that the initial degradation of the sensors' response was not entirely due to the loss in enzyme activity. Non-polarised sensors showed a decrease in sensitivity to glucose and a threshold concentration ca. 3 mM below which they showed no response to glucose, when they were removed from the culture. These effects were not seen before the sensors were placed in the culture and in protein free media, they were partially reversed over time.

Biomaterials play an important role in tissue engineering, where they are used as scaffolds for the 3D culture of cells, to help the generation of neo tissues in-vitro and achieve superior tissue engraftment and regeneration in-vivo. The work presented in this thesis describes how thermoresponsive particle gels, a class of materials not previously investigated for tissue engineering applications, can find important applications in this field. The main gels developed and studied were the aqueous thermoresponsive particle gels prepared from poly(poly(ethylene glycol) methacrylate ethyl ether) (polyPEGMA246-EE) together with polycaprolactone (PCL) microparticles. The thermoresponsive polymer polyPEGMA246-EE, synthesised by free radical polymerisation, was employed as an adsorbing steric stabiliser for polycaprolactone microparticles prepared by the single emulsion solvent evaporation method. The resulting suspensions exhibited reversible temperature induced gelation based on incipient flocculation, where they switched from being free flowing at temperatures below 19°C to form space filling gels at body temperature (37°C) over periods of ~1 minute. On cooling, the suspensions returned to a fluid state. The viscoelastic properties of the particle gels could be controlled by varying the temperature and composition, enabling these gels to be tailored for specific applications. Using NIH3T3 as a model cell line, PCL/polyPEGMA246-EE particle gels exhibited key characteristics advantageous for the 3D culture of cells. These were mainly the ability to assemble around the cells at temperatures, above the LCST of polyPEGMA246-EE, and the provision of a supportive scaffold with appropriate mechanical properties for growth, along with good cytocompatibility enabling cell spreading and proliferation over extended culture times, as well as the rapid return to a flowable state on cooling allowing for suspension transfer, for cell subculture and harvesting, without the need for enzymes. The latter property would also allow for the injectable delivery of the in-vitro conditioned cell-gel constructs for therapeutic applications. Another variant of thermoresponsive particle gels has also been presented in this thesis. Thermoresponsive magnetic-particle gels were developed from the combination of magnetic polystyrene microparticles and the thermoresponsive polymer polyPEGMA246-EE. These exhibited reversible thermogelling behaviour which allowed for cell encapsulation, while their magnetic sensitivity allowed for cell recovery through simple magnetic particle separation. The novel concept of scaffold deconstruction by temperature, and cell recovery through magnetic-particles separation is significant for applications where a scaffold-free outcome would be desired such as the commercial expansion of therapeutic cells. In this thesis, the preparation and application of first generation biocompatible thermoresponsive particle gels is described. The combination of ease of preparation, the potential for scale-up and positive cell response make thermoresponsive particle gels promising as a new class of materials for applications in cell culture, as supports for tissue growth and in cell delivery systems. The materials developed and studied in this thesis are believed to represent a significant contribution to the fields of biomaterials, drug delivery and tissue engineering.

L'essor des thérapies régénératives au cours des 10 dernières années a entraîné un effort de recherche important, mais l'obtention des cellules souches humaines en quantité suffisante reste cependant encore problématique, notamment concernant les cellules souches mésenchymateuses (CSM). Ces travaux ont donc mis en œuvre une approche à la croisée de la biologie et du génie des procédés afin d'identifier les verrous limitant la croissance des CSM. L'étude des méthodes d'intensification de culture a été entreprise grâce à l'utilisation de microporteurs et d'une plateforme de minibioréacteurs de 200~mL. Puis le développement d'un milieu de culture sans sérum a été testé dans le but de maximiser la croissance cellulaire dans des conditions biochimiques contrôlées. Les CSM humaines en tant que modèle type en thérapie cellulaire ont été démontrées comme extrêmement sensibles aux phases de congélation/décongélation, aux variations de température, à un vieillissement prématuré et nécessitant un milieu de culture complexe riche en facteurs de croissance et d'adhérence. Suite à cette étude, plusieurs écueils pourront être évités lors de la montée en échelle d'un procédé de culture de CSM afin d'intégrer leurs paramètres biologiques intrinsèques aux paramètres d'ingénierie des bioréacteurs (transfert de chaleur, contraintes hydrodynamiques, surface d'adhérence) / Progress in regenerative medicines over the past ten years have led to an important research mobilisation, but obtaining a sufficient amount of human stem cells remains nonetheless problematic, especially for mesenchymal stem cells (MSC). Hence, this work developed an approach coupling biology and process engineering to identify barriers limiting MSC growth. The study of scaled-up amplification methods was performed using microcarriers and a 200~mL minibioreactors platform. In order to maximise MSC growth in a biochemically controlled environment, a serum free medium development was tested as well. Human MSC as model cell type for cellular therapies have thus been demonstrated as extremely sensitive to freeze/thaw cycles, temperature variations, subject to premature aging and needing a complex medium enriched in multiple growth and adherence factors. Following this study, several pitfalls might be avoided during MSC process scale-up by integrating the cells biology into the bioreactors' process engineering parameters (heat transfer, hydrodamic stress, adhesion surface)

Ce travail étudie l'intérêt de l'ajout d'hydrolysats de levure dans un procédé de culture de cellules CHO productrices d'anticorps en vue, d'une part, de déterminer leur condition d'utilisation et leur rôle, et, d'autre part, de caractériser les composés actifs. Pour répondre à ces objectifs, une démarche intégrant des études cinétiques, des stratégies de fractionnement et l'analyse biochimique des hydrolysats et de leurs fractions a été développée. En premier lieu, il a été montré que les hydrolysats de levure présentent des effets significatifs sur les cultures selon leur composition et les conditions d'ajout. De même, des effets synergiques ont été mis en évidence par le mélange d'hydrolysats générés à partir de différents procédés. D'autre part, des études cinétiques ont permis de corréler l'influence positive des hydrolysats sur la croissance cellulaire à l'amélioration du métabolisme énergétique. Dans un deuxième temps, la nature biochimique et le rôle des composés actifs ont été étudiés par la mise en oeuvre d'un procédé de nanofiltration membranaire et la reconstitution de mélanges de molécules contenues dans un extrait de levure (EXL). Ces résultats ont mis en évidence l'intérêt des di- et tri-peptides pour approvisionner le métabolisme énergétique et de molécules non nutritives, de poids moléculaire supérieur à 500 Da, pour stimuler la vitesse spécifique de croissance des cellules. Finalement, le rétentat issu de la nanofiltration de l'EXL a été fractionné à l'aide de divers procédés chromatographiques, unitaires ou associés, pour caractériser les propriétés physico-chimiques des composés actifs. L'effet des fractions sur la culture de cellules a alors souligné l'intérêt des molécules chargées positivement, et plus particulièrement, des peptides hydrophiles et cationiques pour stimuler la croissance des cellules. Ainsi, nos travaux permettent de mieux appréhender les mécanismes d'action des hydrolysats de levure sur les cellules CHO productrices d'anticorps et proposent des voies d'optimisation pour la simplification d'additifs complexes dans les milieux de culture dédiés à la culture de cellules animales / This work studies the interest of the addition of yeast hydrolysates in culture medium of CHO cell producing antibody, to determine the operating conditions and their role, but also to improve the characterization of active compounds. In this way, an integrated approach including kinetic studies, fractionation strategies and biochemical analysis of hydrolysates and of their fractions was developed. First, we showed that yeast hydrolysates exhibited various properties depending on their composition and the operating conditions. In addition, synergistic effects were observed with different hydrolysate mixtures. Besides, kinetic studies underlined that the positive influence of hydrolysates on cell growth is correlated with energetic metabolism improvement. Then, the biochemical nature and the role of active compounds were studied by the implementation of a nanofiltration process and the reconstitution of mixtures of molecules contained in a yeast extract (YE). The results highlighted the interest of di- and tri-peptides to supply energetic metabolism, and of non-nutritive molecules, exhibiting a molecular weight greater than 500 Da, to stimulate the specific cell growth rate. Finally, the retentate fraction of nanofiltrated YE was fractionated by various chromatographic processes to characterize the physico-chemical properties of active compounds. The effect of fractions on cell culture emphasized the positive effect of positively charged molecules, especially hydrophilic and cationic peptides, to stimulate the cell growth. Thus, our work provides important insights in yeast hydrolysate mechanisms on CHO cells and suggests procedures to simplify such a complex additive of media dedicated to mammalian cell culture