A High Throughput Manufacturing Technique of Hydrogel Based Droplets for Cell Culturing

This thesis introduces a high-throughput manufacturing technique for producing hydrogel-based droplets developed to address long-standing challenges in three-dimensional (3D) and two dimensional (2D) cell culturing processes. Conventional approaches often struggle to replicate physiologically relevant environments and are limited in terms of scalability, uniformity, reproducibility and stress-free cell culturing. The method presented here uses an automated fiber dipping system to generate consistent and shear-free droplets that encapsulate cells within a supportive hydrogel matrix. Each droplet is immediately stabilized through an ironic crosslinking creating a gentle and reproducible environment for cell growth while allowing precise control over droplet size and structure. The hydrogel system is composed of sodium alginate, carboxymethyl cellulose (CMC), and TEMPO-oxidized nano-fibrillated cellulose (TO-NFC), bio- materials selected for their biocompatibility and tunable mechanical properties. Rheological analyses revealed predictable shear-thinning behavior across compositions making them ideal for droplet formation through dip coating. These behaviors were closely studied through flow curve tests, thixotropic recovery, and frequency sweeps. Substrate interactions were also explored using borosilicate glass and stainless steel with surface energy and contact angle measurements guiding the optimization of droplet retention and spreading. Theoretical models such as capillary number scaling, Rayleigh-Plateau instability, and Tate’s law were used to predict droplet behavior and matched well with experimental outcomes to see the replication. Compositions with intermediate consistency, particularly those with 0.2 percent TO-NFC, showed strong mechanical stability alongside high biological activity. To evaluate cell proliferation within the droplets, the microalgae Auxenochlorella pyrenoidosa was cultured in the hydrogel formulations. Over a seven-day period, growth was monitored using high-resolution microscopy and quantified through image analysis with ImageJ and YOLOv8 based object detection. The results in the droplets formed showed over 80 percent increased cell proliferation in the optimized formulations, significantly outperforming conventional control media. Making it a promising platform for applications in regenerative medicine, tissue engineering, and bio-manufacturing, providing a practical solution for uniform, high-quality 3D cell culture.