preliminary studies

The main objective of this Thesis was to encapsulate single viable cells within polyelectrolyte films using the Layer-by-Layer (LbL) technique. Most of the experiments used human mesenchymal stem cells (MSCs) whose characteristics (capacity of selfrenewal and potential to differentiate into several types of cells) make them particularly interesting to be used in biomedical applications. Also, most of the experiments used alginate (ALG) as the anionic polyelectrolyte and chitosan (CHI) or poly(allylamine hydrochloride) (PAH) as the cationic polyelectrolyte. Hyaluronic acid (HA) was also tested as an anionic polyelectrolyte. At the beginning of the work, the experimental conditions necessary to obtain the encapsulation of individual cells were studied and established. Through fluorescence microscopy visualization by staining the cell nucleus and using polyelectrolytes conjugated to fluorescent dyes, it was possible to prove the obtainment of capsules containing one single cell inside. Capsules aggregation was an observed problem which, despite the efforts to design an experimental process to avoid this situation (namely, by playing with cell concentration and different means of re-suspending and stirring the cells), was not completely overcome. In a second part of the project, single cells were encapsulated within polyelectrolyte layers made of CHI/ALG, PAH/ALG and PAH/HA and their viability was evaluated through the resazurin reduction assay and the Live/Dead assay. In these experiments, during the LbL process, polyelectrolyte solutions were used at a concentration of 1mg/mL based on literature. In general, the viability of the encapsulated cells was shown to be very low/absent. Then, as a consequence of the lack of viability of cells encapsulated within polyelectrolyte layers, the LbL technique was applied in cells growing adherent to the surface of cell culture plates. The cells were cultured like in a sandwich, between the surface of the cell culture dish and the polyelectrolyte layers. Also here, the polyelectrolyte solutions were used at a concentration of 1mg/mL during the LbL process. Surprisingly, cell viability was also absent in these systems. A systematic study (dose-effect study) was performed to evaluate the effect of the concentration of the individual polyelectrolytes (ALG, CHI and PAH were studied) in cell viability. Experiments were performed using cells growing adherent to the surface of cell culture plates. The results pointed out that a very high (cytotoxic) concentration of polyelectrolytes had been in use. Also, in general, PAH was much more cytotoxic than CHI, whereas ALG was the less cytotoxic polyelectrolyte. Finally, using alginate and chitosan solutions with adequate concentrations (low concentrations: 50ng/mL and 1μg/mL), the encapsulation of single viable cells was again attempted. Once again, the encapsulated cells were not shown to be viable. In conclusion, the viability of the encapsulated cells is not only dependent on the cytotoxic characteristics (or combined cytotoxic characteristics) of the polyelectrolytes but it seems that, when detached from the culture plates, the cells become too fragile and lose their viability very easily.Universidade da Madeir