In this thesis, we improved, adapted, and further advanced the CellStudio platform originally introduced in the doctoral thesis of Dr. Enrique Azuaje-Hualde, in which its foundational concept was established. In that work, CellStudio was presented as a proof- of-concept system integrating cell micropatterning with microbead-based functional layers, enabling either localized cellular stimulation or near-cell sensing of secreted factors. Specifically, the solid-phase presentation of fibroblast growth factor 2 (FGF-2) from functionalized microbeads was shown to modulate cell behaviour, including changes in cell number and spatial organization within defined adhesion patterns. In a separate set of experiments, the feasibility of using microbeads as localized biosensors was demonstrated through the detection of cell-secreted vascular endothelial growth factor (VEGF) using antibody-based immunosandwich assays. Importantly,

stimulation and sensing functionalities were validated independently and only in a single cell type, mesenchymal stromal cells (MSCs).

In parallel, the previous thesis introduced an aptamer-based proof-of-concept strategy for VEGF detection in MSC cultures. Although successful in recognizing cell-secreted VEGF, this approach exhibited significant photobleaching under laser excitation, limiting its suitability for dynamic monitoring. In the present thesis, this strategy is expanded through the evaluation of an alternative VEGF-binding aptamer alongside the originally implemented sequence. Comparative analyses were conducted to assess photobleaching reduction, and experimental parameters were systematically optimized to enhance signal stability and imaging performance, thereby advancing the sensing module toward a more robust and application-ready configuration.

While these studies validated the core principles underlying CellStudio, the system remained restricted in terms of biological scope, technical standardization, and functional integration. Building upon this basis, the present work introduces systematic developments aimed at enhancing robustness, reproducibility, and biological validation, while enabling true multifunctionality within the same patterned microenvironment. Specifically, this thesis establishes the simultaneous integration of solid-phase protein presentation and localized biosensing, allowing concurrent control of cellular stimulation and real-time monitoring of secreted factors. Through this integration, CellStudio evolves from an initial demonstration platform into a versatile and modular system capable of addressing more complex and biologically relevant questions.

To demonstrate its adaptability and functional relevance, CellStudio is applied across diverse biological contexts, encompassing multiple cell types and both physiological and pathological conditions. In particular, the platform is employed to investigate cancer- immune cell interactions, cytokine secretion dynamics, and contact-dependent cellular responses, illustrating its utility for immunological assays and tumour-immune communication studies. These applications validate its capacity to dissect the influence of controlled microenvironmental cues on cellular behaviour and position CellStudio as a powerful analytical tool for cancer and immunological research.