2024 AIChE Annual Meeting
Optimizing Hydrogel Microsphere Formation for Suspension Cell Culture
Extracellular vesicles (EVs) are lipid membrane-bound nanoparticles secreted by cells that contain nucleic acids, proteins, and metabolites derived from their cells of origin. While there is much to be learned about their structure and function, they are known to play key roles in intercellular communication and are involved in many physiological and pathological processes. Although EVs have wide therapeutic potential, current GMP that focus on the large-scale biomanufacturing of EVs is very limited. The use of hydrogel microspheres has been recognized as a potential way to scale up the culture of adherent cell lines, and thus the production of EVs. Gelatin methacryloyl (GelMA) is a hydrogel that is UV crosslinkable when mixed with the photoinitiator LAP upon exposure to UV light. Its crosslinked form exhibits robust mechanical properties that provides a 3D structure in which to culture cells at an increased seeding density and for extended culturing time, making it a good candidate for microsphere medium. One proposed method of encapsulating cells consists of a flow-focusing microfluidic chip that facilitates formation of GelMA microspheres through the union of an aqueous phase and an oil phase. Previous optimization efforts studied the ratio of the oil phase to the aqueous and determined that the ratio of the oil phase and gel phase remaining the same but at higher velocities does consistently produce the same-sized spheres but at an increased rate, but spheres are not stable in culture conditions. In an attempt to continue this work and gain further understanding of how to manufacture consistently sized, fully crosslinked, spherical microspheres using this method, parameters such as flow rates, the concentration of hydrogel used, the concentration of LAP photoinitiator added, and oil types were optimized. Microspheres formed under each condition were then characterized by their size, crosslinking efficiency, production, and structural integrity using fluorescent imaging coupled with image quantification and rheological analysis. Moving forward, additional microsphere optimization will be completed after the addition of cells. Ultimately, the goal of this project is to develop a scalable platform to produce EVs from tissue-specific cell types.