Attachment, proliferation and gene expression of anchorage-dependant cells can be influenced by their surroundings, extra-cellular matrix, and the chemistry and morphology of the substrate they adhere to. In-vitro cells are cultured on flat surfaces in a culture flask or a petri dish. In-vivo, they grow next to other cells and tissues, and they are influenced by them and their extra-cellular matrices. In order to study cells in the laboratory, one main focus has been to mimic the natural environment for the cells, as much as possible. One way to increase the similarities between the in-vitro environment and in-vivo, is to replicate micro- and nanoscale surface features or a 3D imprint of cells onto the cell-culture substrates.

This work investigates the fabrication of protein-based biodegradable films as cell-culture substrates, replicated with micro- and nanoscale regular surface features and imprints of cells. Optimisation of these films, which are made of casein (the main protein of cow’s skimmed milk), has also been studied to find the best films according to their flexibility, stability, biocompatibility and the highest obtainable resolution of imprints. The quality of resolution of imprints was tested via atomic force microscopy (AFM) 3D imaging and it was seen that the resolution of features replicated on casein films was closely comparable to the original fixed cells.

Casein is water soluble, thus non cross-linked casein films would dissolve in cell- culture media within a few hours. As a result, in order to use the patterned films as cell-culture substrates, they need to be cross-linked. Cross-linking of casein films with surface patterns, increases their degradation time, thus giving cells enough time to adhere to the films and grow into layers of cells, as directed by the pat- terns, before the films start to degrade. The optimisation process also included cross-linking of the films using different cross-linking reagents and methods. Two and a half-dimensional regular features (2D geometric shapes with a constant depth for all features) were transferred onto casein films using photolithography and soft- lithography. Three-dimensional topography of cellular microenvironments were also replicated onto casein films using a modified bioimprinting method. For both regu- lar features and bioimprints, polydimethylsiloxane (PDMS) moulds, made via soft- lithography, were used as the intermediate mould for liquid-casting casein on, and transferring the features onto casein.

Using liquid-casting casein on PDMS moulds in the replication process, the res- olution of features on casein films was poor, compared to the original features on photoresist, the original cells, and the imprints on PDMS. PDMS is hydrophobic by nature and despite of plasma treatment, it only remained hydrophilic for a short period of time. Hence casein solution, being water-based, could not wet the surface well enough and get completely into the micro- and nanoscale details on PDMS. As a result, the fabrication process was optimized, and PDMS moulds were treated via oxygen plasma and polyvinylpyrrolidone (PVP), prior to liquid-casting of casein. PVP is a water-soluble hydrophilic polymer, which binds to the surface of PDMS and renders it hydrophilic for a much longer period of time. Addition of this step to the replication process, helped with casein solution filling the details on PDMS better. This led to high resolution patterns on the final casein films. Optical images and AFM images were taken of regular features and bioimprints in order to compare the features at different stages of replication.

It was found that casein films made of 15%(w/w) casein in 0.2%(w/v) NaOH solution, mixed with 15% (w/w) glycerol as plasticizer, fitted best within the scope of this work. These films were cross-linked by mixing the casein solution with transglutaminase (TG) prior to liquid-casting on PDMS moulds. Concentration of TG in the solution was 10 U per gram of protein. These films were patterned with regular features and bioimprints, and patterned films were successfully used as cell-culture substrates.

The results reported in this thesis provide a foundation for potential research and commercial applications for biodegradable cell-culture substrates or implants with surface features.