Engineering scalable exosome isolation platforms and biomimetic scaffolds for ADSC-based regenerative therapies

Abstract

The therapeutic use of exosomes, small extracellular vesicles involved in paracrine signaling and intercellular communication, has emerged as a promising alternative to cell-based therapies in regenerative medicine. However, their clinical application remains limited by challenges in scalable production, efficient purification, and functional validation in biologically relevant models. This dissertation addresses these limitations by integrating bioprocess optimization with engineered human-based platforms for tissue regeneration. The experimental work presented in this thesis is divided into two main parts. The first part explores strategies for the scalable and selective isolation of exosomes from mammalian cell cultures. This included the design and optimization of aqueous two-phase systems (ATPS) for the purification of CaCo2-derived exosomes, which achieved high recovery efficiency (>80%) with minimal protein contamination. A comprehensive review of emerging technologies such as microfluidics, membranebased methods, and bioreactor platforms was also conducted and classified according to their scalability and purity output. The second part investigates the regenerative and immunomodulatory potential of ADSC and their exosomes using advanced biomimetic systems. A human in vitro burn wound model was developed to assess the impact of ADSC-derived exosomes on macrophage modulation and angiogenesis, resulting in enhanced vascularization and the immunomodulatory regulation of IL-6 and IL-10 expression. In parallel, a set of tunable ternary scaffolds composed of collagen, elastin, and fibrin were developed to support adipose tissue regeneration both in vitro and in vivo. The scaffolds enhanced the adipogenic differentiation of ADSCs without external induction, as confirmed by the upregulation of adipogenic marker such as CEBPα, PPARγ, FABP4 and Caveolin-1. By combining scalable manufacturing strategies with functional evaluation in biomimetic systems, this dissertation contributes to the development of next-generation platforms for exosome-based regenerative therapies. The findings presented here offer new insights into the engineering of both bioprocesses and bioactive scaffolds, supporting future applications in personalized medicine and soft tissue repair