Poster M13 - High-throughput bioprinting of cell-laden droplets for biomanufacturing of organoids

Introduction: The thymus is the primary lymphoid organ where T cell precursors develop and mature into T cells that are critical to immune functions. Impaired thymic functions lead to life-threatening immunodeficient conditions. Transplantation of thymic organoids derived from human induced pluripotent stem cells (hiPSCs) has been proposed as a promising way to restore individualized thymic functions. However, the process of generating consistent and robust derivations of mature organoids from hiPSCs is challenging. Activation of cellular mechano-transduction pathways has been proposed to overcome this challenge. In this study, we demonstrate a robust bioprinting technique that generates cell-laden alginate droplets with tunability in size and stiffness suitable for engaging mechano-transduction pathways.

Materials and

Methods: We developed a semi-automated high-throughput bioprinting system that produces cell-laden droplets for biomanufacturing of organoids. The bioprinting system 1) encapsulates cells into alginate droplets using air-jetting technology and 2) performs automated wash cycles following droplet generation. A Raspberry Pi single-board computer was used to interface and control the syringe pump for droplet generation and the peristaltic pumps for wash cycles. We developed a custom Python script to automate the printing and washing sequence with minimum human intervention, improving efficiency and reducing the risk of contamination. We conducted comprehensive studies to characterize the size and shape of the droplets generated under various printing conditions and bioink formulations. Additionally, we quantified the droplet production rate using a high-speed camera. Following bioprinting, cell-laden droplets were cultured for aggregation and differentiation.

Results, Conclusions, and Discussions: Our study showed that a higher airflow rate (6L/min v.s. 4L/min) generated smaller alginate droplets and that a higher CaCl2 concentration (200mM v.s. 100mM) crosslinked alginate produces more spherical droplets. Furthermore, increasing the alginate concentration (2.0% v.s. 1.6%) improved the integrity of the droplets. Through optimization, we developed a robust cell encapsulation protocol that allowed us to reliably generate cell-laden and monodisperse droplets in 400 to 600 µm size range. The droplets can be generated at a rate of approximately 50 droplets per second, which translates into 27,000 droplets in 10 minutes. Cell culture has shown successful cell growth, aggregation, and differentiation with high cell viability. While the cell encapsulation technique was developed for generating thymic organoids in this study, this technique can equally be applied to other types of organoids. Future work will focus on further improving the throughput towards mass biomanufacturing of organoids.

Acknowledgements (Optional): This study was supported in part by the NSF FMSG 2229156 grant sub-awarded to Duquesne University.