Segmented hydrogels: process development of a reproducible 3D tissue engineered interface system and its use as a muscle–tendon model EfendiKyle 2019 Prior to commercialisation, all drugs and medical devices must undergo testing to ensure safety to the end user. Part of this process is the pre-clinical trials stage in which high-throughput testing of the product is performed on cells in monolayer followed by testing in animal models. Monolayer cultures are generally basic, containing one cell type, which leads to minimal testing parameters. The more complex animal tests are often misleading as they do not adequately represent the human physiology and their ethics are also often contested. 3D Tissue engineered models, an evolution of the monolayer model more accurately mimic the structure and biochemistry of specific native tissues. To observe effects on the musculoskeletal system, a model representing these tissues is necessary. This thesis focuses on attempting to create an in vitro myotendinous junction (MTJ) for such purposes. Firstly, the most suitable published process for making a 3D tissue engineered skeletal muscle model was identified based on an analysis of requirements. A model using the C2C12 cell line in a collagen hydrogel between two anchor points was chosen and the process was optimised using a Quality-by-Design framework. This was essential to make a system that would lend itself to high-throughput testing in the long run. Following this, a simple process for creating an MTJ, termed ‘segmentation’ of the gel, was tested and showed a reduction in surface area consistent with cell attachment as previously reported. This involved physically blocking regions of the gel during manufacture. Multiple design iterations were tested to enable reproducibility. Of the tested configurations, a 3D printed PLA mould adhered to a 6-well plate with sliding dividers for segmentation and posts for gel anchor points was found to be optimal. Finally, standardising the use of ice in the gel fabrication process to prevent premature polymerisation of the hydrogel led to the success rate of fabrication to increase to up to 100%. Comparisons with the initial system showed multiple indicators of more consistent gels with reduced failure rates, a reduction in the resources required due to scaling down, and versatility in the design allowing for segmentation and simple adaptation to testing apparatus for future experiments. This system was then tested by only seeding the central region of a gel with C2C12 muscle-precursor cells to create “segmented gels”. Compared to homogenously seeded constructs, the ‘muscle’ region in segmented gels was found to have no difference in macroscopic behaviour and only a slight decrease in myotube width measurements, still within published parameters. These models exhibited a unique ‘bow-tie’ shape from the seeding discrepancies in the different regions. During the 14-day culture period, the cells became equally distributed throughout the gel, indicating that they may be migrating over the culture period. These regions also exhibited myotube formation and although less densely populated, a greater incidence of striated myotubes were found in these regions as demonstrated by staining with rhodamine phalloidin. Finally, the end regions were seeded with human dermal fibroblasts (hDFs) to represent a tendon to create a tendon-muscle-tendon model. Immunostaining showed that the majority of cells in the resulting construct were desmin-positive, a muscle-specific marker. This is in agreement with previous research that shows that dermal fibroblasts can be driven down a myogenic lineage by secreted factors in culture. However, transitional interdigitation between the two morphologically different cell types were observed in some models. This represents the first report of the successful formation of a myotendinous junction in a collagen-based potentially high-throughput system.