Développement d'un système tridimentionnel de culture cellulaire pour orienter la formation de microvaisseaux
SubjectCultures en bioréacteur
The development of functional and oriented microvessels within tissue constructs is a tremendous challenge in tissue engineering and regenerative medicine. It is justified by the need to allow better nutrient and oxygen supply and waste removal within the core of tissue constructs. It is one of the reasons why only few tissue substitutes are available to clinicians. Therefore, the focus of many research studies in the field of tissue engineering has been placed on promoting microvessel ingrowth inside tissue constructs prior to their implantation, as presented in Chapter 1. This process is often referred to as pre-vascularization. As such, Chapter 2 of this thesis is devoted to review the recent development and future challenges related to the microvascularization process of tissue constructs.The experimental work in this thesis is based on the hypothesis that polymer monofilaments precisely distanced from each others can be used to guide endothelial cells when dispersed in a (fibrin) gel and to induce tube-like structures in a directional fashion. In Chapter 3 of this thesis, the use of polymer fibres as contact guidance of endothelial cells to orient microvessel formation and development in a three-dimensional environment was validated.The novel cell attachment method described in Chapter 3 has resulted in an increase in Human Umbilical Vein Endothelial Cell (HUVEC) adhesion and spreading on bare poly(ethylene terephthalate) (PET) fibres. Furthermore, HUVEC-covered fibres were sandwiched between fibrin gels to allow the development of microvessels. Angiogenesis development was characterized and evaluated using a number of imaging techniques, including fluorescence microscopy and confocal microscopy. After 4 days of culture, microvessels formed large tube-like structures (diameter of approximately 100 [micro]m) between adjacent fibres distanced by 0.1mm. This proof-of-concept opens the door to other experiments and to possibility of scaling the system to bioreactor cultures. In Chapter 4, the effect of human fibroblasts and in another set of experiments, the influence of two angiogenic growth factors (i.e., Vascular Endothelial Growth Factor (VEGF) and basic Fibroblast Growth Factor (bFGF)) over the responses of the HUVEC-covered fibres sandwiched in fibrin were investigated.The development and maturation of microvessels as well as the assessment of lumen formation were evaluated using a fluorescent fibrin matrix, confocal microscopy and histology. Histology and fluorescent fibrin experiments confirmed that these microvessel-like structures formed between polymer monofilaments embedded in fibrin contained a lumen.The effects of VEGF and bFGF were dose dependent. Furthermore, the use of fibroblasts significantly improved the maturation of the microvessels compared to control and to samples cultured with VEGF and bFGF. Conclusions and suggested future work are presented in Chapter 5. Appendices A and B present the detailed protocols used to label cells in this study and general information about cell cytoskeleton, respectively.
- Génie – Thèses