Characterization of casein-based films for cell-culture and medical implants

Abstract

The objective of this thesis was to optimize casein-based biodegradable films for use as cell culture substrates and medical implants. These films were patterned with 3D imprints of cells to influence cell phenotype. Secondary cells align on surface patterns and dissolve the material in the process. In order to find the best films, characterization of these films was made according to their biocompatibility, flexibility, stability and high resolution of imprints. More specifically, the characterization of different casein-based films was studied as a function of protein and glycerol concentration as well as the type and the concentration of crosslinking reagents. Films were crosslinked with glutaraldehyde, formaldehyde, citric acid and transglutaminase. Mechanical properties such as stiffness and glass transition were characterized using Dynamic Mechanical Analysis (DMA). Spectrophotometry was used to measure the transmittance of the films and an epifluorescence microscope at 595 nm was used to analyzed the film autofluorescence. Contact angle, degradation time and water uptake ratio were also investigated in DI-water and in media at 37.5°C in order to mimic the cell environment. An increase of glycerol concentration level increased the transmittance of the film. However, an increase of casein concentration level decreased the transmittance. On the other hand, glutaraldehyde and citric acid films had an orange-brown color and did not transmit light under 400 nm. Casein-based films were not enough autofluorescent to influence cell analysis using fluorescent dyes or labels except citric acid and glutaraldehyde-crosslinked films. Indeed, the two latter films had saturated fluorophores with an exposure time of 2 seconds. An unexpected result was observed regarding to contact angle. TG-crosslinked film had the highest contact angle and was the only cell-friendly film. Contact angle of the film did not influence the cell adhesion. Degradation time of the films varied according to the crosslinker concentration. Higher the crosslinker concentration level was, higher the degradation time was. In addition, degradation time of films immersed in media was longer than films immersed in DI-water. Swelling of the films decreased with the increase of crosslinker concentration level. Formaldehyde and citric acid films had a low water uptake ratio. TG-crosslinked casein-based film had a very high water uptake ratio in DI-water but not in media. Diameter expansion and patterning expansion of crosslinked films were also investigated. In a logical way, their expansion was lower in media. An optimal film for use as cell-culture substrate could be deducted from these results. Casein-based film with 10 U/g of transglutaminase was transparent, slightly fluorescent, biocompatible and did not expand much in media (about 30\%). All of these characteristics are favorable for this application. Indeed, standard microscope can be used for cell analysis if the material is transparent. A biocompatible material induces a good cells adhesion. If the material does not absorb a lot of water, bioimprints are similar to the cultured cells throughout the process and cells might differentiate. Another aspect of this project was aiming to develop 3D structures of casein by mixing casein solution with gelatin to add gelation properties. Gelatin/casein films were transparent, slightly autofluorescent and did not absorb a lot of water. They are promising for use as cell-culture substrates and medical implants.