Skeletal Muscle Differentiation in 3D Capillary Gels
Examensarbete för masterexamen
Skeletal muscle tissue contributes to many functions in the human body such as locomotion and maintaining the body temperature. Tissue engineering could open up possibilities of grafting muscle tissue ex vivo and make way for new autologous treatments mitigating pathological muscle loss. Additionally, skeletal muscle tissue of mammal origin is one of the main nutritional protein sources in the western diet. Also here the production of skeletal muscle tissue ex vivo could provide measures to reduce environmental strains due to conventional live-stock production and produce food in a more resource efficient manner. Until a market maturity is achieved, methods have to be found to produce cells in high enough numbers and the differentiation has to be ensured to allow for functional tissue for medical applications and nutritionally valuable food. Additionally, the production of larger tissue-engineered constructs is challenging due to the absence of a perfusion network bringing the nutrients and oxygen deep into the constructs. The aim of the project is to grow parallel aligned muscle fibres in a three dimensional scaffold with the help of a perfusion bioreactor. The approach was to grow and differentiate C2C12 mouse myogenic progenitor cells inside capillary alginate gels. It was hypothesized that the parallel aligned capillary structure inside the gels could help the alignment of the muscle fibres and prospectively be used in generating muscle constructs closer to physiological muscle found in mammals. In order to promote cell attachment to the gels, different modifications of the alginate gel have been performed. Bulk modifications have been achieved by mixing pure alginate solutions with pre-coupled RGD-alginate or gelatin solutions prior to crosslinking. Furthermore, surface modifications have been performed via collagen coatings or carbodiimide coupling of RGD peptides to the surfaces of crosslinked alginate gels. Cell cultures were performed on the surface of samples cut from these gels to evaluate the attachment improvement of the various modifications and to study the influences of the capillaries on alignment of the cells. In addition to the cultures the scaffolds have been investigated by light and confocal microscopy. For three dimensional cell cultures a perfusion bioreactor has been designed. Computer-based simulations have been performed for in silica evaluations of the flow and oxygen distribution inside the bioreactor and the capillaries of the alginate gels. Eventually, the bioreactor and the RGD:alginate bulk modified gels were combined in 3D culture experiments to characterize the culture setup in different operational modes, to formulate protocols and to test the feasibility of the experimental setup for cell growth and differentiation. As a result of this project, a bioreactor system has been developed allowing for future investigations of capillary alginate gels as a culturing scaffold for skeletal muscle progenitor cells. The bulk modification with RGD-alginate seems to be most beneficial in growing high cell numbers and achieving good attachment quality of the cells to the alginate gels. Optimization of the sterilization techniques in concert with improved modifications of the alginate scaffold to better present the functional groups to the cells could lead to more promising results of the cell cultures. CaCl2 is recommended to be replaced by another crosslinking agent because the gels crosslinked with Ca2+ do not have the necessary structural integrity throughout the cell culture. A switch to covalently bound gels and more careful tailoring of the scaffold properties, e.g. elastic modulus, stress relaxation behaviour, could improve the outcome of the cell cultures. This project serves as a basis for future advancements in culturing skeletal muscle cells in vitro for nutritional as well as medical applications.
Livsvetenskaper , Grundläggande vetenskaper , Hållbar utveckling , Innovation och entreprenörskap (nyttiggörande) , Klinisk medicin , Life Science , Basic Sciences , Sustainable Development , Innovation & Entrepreneurship , Clinical Medicine