Bénédicte FROMAGER
defended her PhD thesis on 15 december 2023
Membrane of electrospun polyacrylonitrile fibres for 3D cell culture
Front of the jury composed of:
– Pascale CHENEVIER,DrHDR,SyMMES,CEAGrenoble – Rapporteure
– Valérie CORONAS, Pr,STIM,Université de Poitiers – Rapporteure
– Cécile FEUILLIE,DrCRCNRS,CBMN,Université de Bordeaux – Examinatrice
– Mikhael BECHELANY, DRCNRS,IEM,Université de Montpellier – Examinateur
– Julien CAMBEDOUZOU, Pr, IEM,Université de Montpellier – Directeur de thèse
– David CORNU, Pr, IEM,Université de Montpellier – Directeur de thèse
– Norbert BAKALARA,Pr,CBMN,ENSTBB, Bordeaux – Invité
– Emilie MARHUENDA, Dr, Queen Mary University of London – Invitée
Abstract:
Cell behaviour depends on the composition and organisation of the extracellular matrix (ECM). Obtaining a biomimetic environment of the ECM for cell growth therefore represents a challenge that could open up unprecedented prospects. Among the existing 3D cell culture supports, membranes obtained by electrospinning offer very interesting advantages, in particular a fibrillar system mimicking that of the ECM. In this context, the collaboration established between the IEM and the INM has led to the design of 3D membranes composed of electrospun ex-PAN (polyacrylonitrile) fibres for cell culture. These membranes are biocompatible, can be inserted into the wells of cell culture plates, and do not degrade under the effect of cell culture products or in the time scale of measurements. In addition, it is possible to study the impact of mechanical and physicochemical properties independently. In a previous thesis, these membranes were used to study the migration of glioblastoma cells (GSCs), which forms the basis of this work. In this manuscript, the first chapter is devoted to a presentation of electrospinning and the state of the art in the use of membranes obtained using this method for cell biology. The second chapter describes the systematic and rational process for characterising the topography and porosity of the membrane using DiameterJ (Image J software plugin). In addition, we ensured that the introduction of carbon nanotubes into the initial solution, to vary the mechanical properties of the fibres, had no impact on the topography of the membrane. Various analyses (TEM, nanotomography) led to the conclusion that the CNTs were intrafibrillar. Chapter 3 presents the characterisation of the membrane topography, the surface functionalisation and the specification of the mechanical properties. In order to obtain a biomimetic support, we attempted to covalently functionalise the membranes with RGD motifs. We then attempted to measure stiffness as a function of CNT content using AFM and nanoindentation. The trend observed was an increase in stiffness as the filler content increased. The influence of these properties on A7R5, C2C12, NRC, iPSCs, GBM and MCF7 cells is then studied in chapter 4, after cytotoxicity tests to ensure the biocompatibility of the support. This study showed that the cells infiltrate the support, proliferate and their behaviour is impacted by the topography and mechanical properties. The ‘outlook’ section presents the preliminary results obtained on two subjects. The first concerns the ability to obtain biomolecules of pharmaceutical interest in a bioproduction process. The second concerns the development of Physarum polycephalum, an organism that could be grown on our supports as a model system, particularly for amoeboid migration and network development.
Cell behaviour depends on the composition and organisation of the extracellular matrix (ECM). Obtaining a biomimetic environment of the ECM for cell growth therefore represents a challenge that could open up unprecedented prospects. Among the existing 3D cell culture supports, membranes obtained by electrospinning offer very interesting advantages, in particular a fibrillar system mimicking that of the ECM. In this context, the collaboration established between the IEM and the INM has led to the design of 3D membranes composed of electrospun ex-PAN (polyacrylonitrile) fibres for cell culture. These membranes are biocompatible, can be inserted into the wells of cell culture plates, and do not degrade under the effect of cell culture products or in the time scale of measurements. In addition, it is possible to study the impact of mechanical and physicochemical properties independently. In a previous thesis, these membranes were used to study the migration of glioblastoma cells (GSCs), which forms the basis of this work. In this manuscript, the first chapter is devoted to a presentation of electrospinning and the state of the art in the use of membranes obtained using this method for cell biology. The second chapter describes the systematic and rational process for characterising the topography and porosity of the membrane using DiameterJ (Image J software plugin). In addition, we ensured that the introduction of carbon nanotubes into the initial solution, to vary the mechanical properties of the fibres, had no impact on the topography of the membrane. Various analyses (TEM, nanotomography) led to the conclusion that the CNTs were intrafibrillar. Chapter 3 presents the characterisation of the membrane topography, the surface functionalisation and the specification of the mechanical properties. In order to obtain a biomimetic support, we attempted to covalently functionalise the membranes with RGD motifs. We then attempted to measure stiffness as a function of CNT content using AFM and nanoindentation. The trend observed was an increase in stiffness as the filler content increased. The influence of these properties on A7R5, C2C12, NRC, iPSCs, GBM and MCF7 cells is then studied in chapter 4, after cytotoxicity tests to ensure the biocompatibility of the support. This study showed that the cells infiltrate the support, proliferate and their behaviour is impacted by the topography and mechanical properties. The ‘outlook’ section presents the preliminary results obtained on two subjects. The first concerns the ability to obtain biomolecules of pharmaceutical interest in a bioproduction process. The second concerns the development of Physarum polycephalum, an organism that could be grown on our supports as a model system, particularly for amoeboid migration and network development.
Résumé grand public :
In order to obtain relevant results in cell biology, a matrix that mimics the extracellular matrix (ECM) is essential. During my thesis, an in-depth characterization of a bio-inspired and biomimetic electrospun fibre matrix was carried out. I established an image analysis method to obtain quantitative information on the topography and porosity of the support. Carbon nanotubes were introduced into the initial solution to modulate the mechanical properties without impacting the overall structure of the membrane. We also assessed these properties and functionalized them with ECM proteins. After verifying biocompatibility, the impact of the support properties on cellular behavior was assessed. Finally, preliminary results are presented for two applications: obtaining biomolecules in a bioproduction process and growing Physarum polycephalum, a potential model for cell migration.
In order to obtain relevant results in cell biology, a matrix that mimics the extracellular matrix (ECM) is essential. During my thesis, an in-depth characterization of a bio-inspired and biomimetic electrospun fibre matrix was carried out. I established an image analysis method to obtain quantitative information on the topography and porosity of the support. Carbon nanotubes were introduced into the initial solution to modulate the mechanical properties without impacting the overall structure of the membrane. We also assessed these properties and functionalized them with ECM proteins. After verifying biocompatibility, the impact of the support properties on cellular behavior was assessed. Finally, preliminary results are presented for two applications: obtaining biomolecules in a bioproduction process and growing Physarum polycephalum, a potential model for cell migration.
PhD defense: Bénédicte FROMAGER – 15/12/2023
