Airflow controlled droplet bioprinting enables high-throughput generation of iPSC-derived thymic organoids with in vivo T cell functionality.

The thymus is a critical organ for T cell generation. It is composed of distinct layers of thymic epithelial cells (TECs) that coordinate a complex process of antigen recognition and self-tolerance checks that result in mature populations of T cells. However, it undergoes progressive and permanent involution and loss of functionality which peaks in adolescence, rendering the tissue largely non-functional. Induced pluripotent stem cells (iPSCs) have emerged as promising candidates to generate organoid models of the thymus because of their autologous potential and indefinite self-renewal. Previously our group has shown the capability of generating thymic epithelial progenitor cells (TEPCs) that are able to successfully program T cells in vitro and in vivo through manual encapsulation of iPSCs in sodium-alginate capsules during differentiation. To translate this capability toward clinically relevant throughput, we present a novel method of air-jetting droplet bioprinting which is capable of producing 21,500 aggregates per milliliter of bioink. We conducted a systematic analysis of critical process parameters governing iPSC aggregation, retention, and differentiation and illustrated the interplay between airflow velocity, which governs capsule size, and crosslinker chemistry governing capsule network stability. We also determined parameters that minimize printing variability and prevent decapsulation of iPSC aggregates during differentiation. Finally, we modelled the growth rate and carrying capacity of different printing configurations, and validated manufactured organoids in vivo by generating functional murine T cell populations in athymic nude mice after decapsulation and construction with a decellularized thymus scaffold.