(68f) Development of a High-Throughput Microgel Platform for Cancer Micro-Organoid Generation and Drug Screening Via Pipetting

Advancements in disease and therapeutic response modeling hold significant promise for enhancing patient treatment strategies and developing more effective therapies. Traditional 2D modeling platforms often fall short in replicating key physiological aspects of native tissue microenvironments, such as cell-cell interactions, proliferation dynamics, mechanical stimuli, and tissue development. Consequently, there is a pressing need for novel in vitro platforms capable of simulating diverse pathophysiological pathways to enhance disease modeling and therapeutic discovery, particularly in the context of cancer.

Cancer multi-cellular constructs and organoids offer valuable platforms for modeling tumor growth and drug screening by preserving physiologically relevant aspects of native tumor microenvironments. However, their potential to guide clinical decisions is impeded by challenges such as limited organoid production time and variability in reproducibility. To address this issue, we have developed a home-made pipette microfluidic device for the high-quantity generation of cancer micro-organoids via cell encapsulation by droplet emulsion for high-throughput drug screening. We synthesized norbornene-modified hyaluronic acid (NorHA) polymers, which serve as extracellular matrix (ECM) mimics, to create a suitable cell-encapsulation platform. The viscosity of the polymer solution was adjusted to enable its extrusion by a home-made elliptical pipette for droplet generation with suspended ovarian (OVCAR 5) and prostate (LNCaP) cancer cells (6x10⁶ cells/mL) via water-in-oil (W/O) emulsification.

We achieved the rapid generation of thousands of monodispersed droplets ranging from 370 µm to 420 µm via covalent crosslinking by thiol-Nor complexes, which allowed for further emulsion breaking and separation of gel droplets with ~80% separation efficiency. RGD motifs were added to the gel formulation to improve cell adhesion. A PDMS microfluidic device featuring microwells of 450 µm in diameter was fabricated to immobilize individual microgels to monitor micro-organoids growth via microscopy analysis and cell proliferation assays. Combinations of non-degradable (DTT) and degradable (MMP-sensitive) motifs were used as crosslinkers to optimize cell proliferation rate. Cell viability assays were conducted upon droplet separation on day 1 and 8 under culture. The initial viability for both the OVCAR and LNCaP cell lines was ~77%, which increased up to 90% for day 8 due to cell proliferation. The ability of small molecules to permeate the microgels was assessed using TRITC-labeled dextran (4400 Da), which showed a significant internalization and accumulation of the fluorophore at 30 min and 60 min, respectively. Finally, anticancer drug screening was performed via WST-1 and luminescence assays using doxorubicin (DOX) to model the sensitivity of the micro-organoids towards therapies administration. The estimated IC50 was ~0.04 µM and ~0.3 µM for the LNCaP and the OVCAR5 micro-organoids, respectively.

Overall, the developed microgels platform showed suitability for the encapsulation of different cancer cell lines and organoids' growth, which would allow the screening of multiple tumor microenvironment conditions. The versatility of the technology allows it to be scaled up with multi-channel and/or robotic systems and integrated with flow cytometry to realize a high-throughput platform for patient-derived micro-organoids generation and drug screening.