(634a) Scalable and Reproducible Biomanufacturing of iPSC-Derived Organoids

While significant progress has been made in developing and optimizing protocols for induced pluripotent stem cell (iPSC) organoid generation, research is still limited in developing technologies to approximately scale the production of these organoids. With the increased interest in using organoids for drug discovery, disease modeling, and tissue engineering, the demand for organoids has risen, whose production is largely dependent on the 2D or 3D environment they are cultured in. iPSCs add another layer of challenge, as they are very sensitive to changes in their environment, resulting in heterogenous and low volume differentiation. To address the problem of scale-up for organoid generation, we propose an efficient, low-cost iPSC organoid generation system that provides the correct biophysical cues for a successful and reproducible differentiation.

In a collaborative effort, an air jacket droplet bioprinting system was developed to facilitate the scalable derivation of iPSC-organoids. Alginate bioink was extruded from a syringe pump into a crosslinking bath, with an air jacket at the outlet, creating microscale beads. The size of the bead could be controlled by the air flow rate supplied, and the concentration of alginate printed. These microscale alginate droplets mimic the microscale structure of tissue and can provide the correct biophysical cues for differentiation.

To start, the alginate concentration, air flow rate, and ink extrusion rate were optimized to form high volume, uniform, blank microscale beads. These beads were generated from 2.0% w/v alginate and crosslinked with 100 mM CaCl₂, resulting in 42 500-μm beads produced per second of printing. Next, we examined whether printed single iPSCs could aggregate within the printed beads, a necessary requirement for downstream differentiation. iPSCs were seeded at 1 million cells: 1 mL alginate, printed and crosslinked in 100 mM CaCl₂. Beads were cultured for 4 days, where clear aggregate formation was noted by Day 2, with an average aggregate size of 46 μm noted by Day 4. Aggregates were both viable and pluripotent, demonstrating excellent expression of OCT4 and NANOG. In terms of scaling, on average 6 aggregates were generated per 500 μm bead, resulting in an aggregate production rate of 250 aggregates per second of printing.

As a proof of concept for scaling of differentiation, cells embedded in the beads were differentiated over 11 days to an iPSC-derived thymus epithelial progenitor cell (TEPC) organoid. Aggregates in the beads reached an average size of 114 μm, but decapsulated at a significant rate starting Day 5 of differentiation, which reduced the average aggregate number per bead to 1.33. Although the aggregate number was reduced, differentiation was still successful, with organoids displaying expression of KRT8 and EpCAM, key TEPC markers. While crosslinking beads in CaCl₂ and differentiating successfully produced TEPC organoids, the relative malleability of the crosslinked beads led to this significant decapsulation, resulting in a loss of product.

We next explored the effect of changing the bead stiffness in regard to cell decapsulation rate and expression characteristics, in order to mitigate the loss of production. Beads were printed and crosslinked in either 35 mM BaCl₂, 70 mM SrCl₂, or 200 mM CaCl₂. In all crosslinking conditions, single cell iPSCs were able to successfully aggregate, and mature into iPSC-derived TEPC organoids. High cell viability was noted in all conditions, and key TEPC phenotypic expression was induced in the cells, with 68% of cells EpCAM⁺ in the Ca crosslinked beads, 62% of cells EpCAM⁺ in the Sr crosslinked beads, and 84% of cells EpCAM⁺ in the Ba crosslinked beads. By increasing the bead stiffness through increasing the cation strength (Ba > Sr > Ca), the number of aggregates decapsulating from the beads was significantly reduced, resulting in virtually no decapsulation seen in Ba crosslinked beads.

On average when cell embedded beads were printed, crosslinked in 35 mM BaCl₂, and cultured to the iPSC-derived TEPC lineage, 21,500 ± 5750 TEPC aggregates were generated per mL of bioink. This further resulted in a 22.8 ± 6.1 cell number fold increase from the starting cell density. This significant increase in cell production combined with the high viability and phenotypic expression noted after differentiation, indicates that this droplet bioprinting system has the potential to meet the quality and volume scale needed for iPSC-derived organoid production in a clinical research setting. Work is currently ongoing to see if TEPC organoids generated within the bead can be implanted and induce T cell maturation, ensuring that the cells produced are also functional.