(275b) Biomimetic Hydrogel-Based Platform for Engineered Organoid Culture and High-Content Drug Testing

High-content and high-throughput screening (HCHTS) with enhanced drug testing efficiency have drawn great attention in the field of drug discovery and drug development. Yet, traditional 2D HCHTS in well-plates often diverge from in vivo studies, hindering drug development due to the lack of complexity in living organisms. Three-dimensional (3D) cell cultures, such as organoids, offer a promising solution by mimicking in vivo conditions, replicating tissue structures and functions more accurately, and facilitating reliable cellular behavior and drug response. However, persistent challenges in organoid-based 3D HCHTS impede widespread adoption, including scalability issues and limitations in organoid size distribution, leading to a trade-off between throughput and uniformity. These challenges jeopardize system reliability, particularly in molecular transport and nutrient/drug distribution, which is crucial for accurate drug effect estimation. Integrating organoids into HCHTS requires additional strategies that may disrupt simple workflow design and lack compatibility with conventional readouts, potentially hindering the original goal of enhancing drug testing efficiency. Hence, it still remains crucial to implement 3D HCHTS effectively, ensuring (1) consistent and uniform organoid formation, (2) reliable systemic function with consistent molecular and drug transport, and (3) easy compatibility with conventional imaging and analysis techniques for HCHTS.

This research presents an agarose hydrogel-based scaffold featuring an inverted colloidal crystal (ICC) geometry, specifically engineered for consistent and high-yield spheroid/organoid cultivation (Figure 1A). Uniform-sized microgels derived from various alginate concentrations through the electrospraying technique (Figure 1B) serve as the primary framework for the ICC scaffold. Notably, the alginate content influences the mechanical stiffness of the microgels, particularly their compressive moduli (Figure 1C). On the other hand, the exceptional uniformity of these microgels induces their crystalline assembly, eventually resulting in hexagonal crystal packing (HCP) domains under simple sonication within 1 min (Figure 1D). Here, their compressive moduli promote their overlap when they are packed, culminating in the formation of uniform spherical caps (Figure 1E). Subsequently, casting low-melt agarose hydrogel above its melting temperature successfully encapsulates the microgel assembly without affecting the HCP domain of microgels and their overlapping caps. In final, the enzymatical and chemical degradation of microgels inside results in the agarose-based ICC scaffold featuring HCP domains with uniform void spaces (Figure 1F), multiple layers (Figure 1G), and interconnecting channels (Figure 1H).

With its outstanding biocompatibility, customizable nature, and ability to replicate intricate cell-to-cell interactions, the ICC scaffold is ideal for generating in-vitro models as high-content organoids relevant to tissue engineering and disease simulation (Figure 1I). The scaffold exhibited both cost-effectiveness and safety for large-scale organoid cultivation across various cell lines, achieving an impressive yield of over 1000 organoids per well within a standard 96-well plate. Moreover, the unique geometry of the ICC scaffold, characterized by an HCP arrangement, seamlessly integrates with real-time monitoring techniques, significantly advancing our understanding of drug therapeutics through high-content assessments (e.g., >10 organoids per minute; over 300 times faster than traditional organoid analysis methods), enabling the implementation of reliable 3D HCHTS (Figure 1J).

In conclusion, our biomimetic ICC scaffold represents a significant advancement in 3D cell culture, offering promising implications for fields such as tissue engineering, disease modeling, drug development, and biomedical research.