(A-28) Development of a High-Throughput Drug Screening Platform Via Pipetting Gel Droplet Micro-Organoids Models

Introduction:: Disease and therapeutic response modeling has gained significant attention due to its potential to improve patient treatment decision-making and develop more effective therapies. However, 2D modeling platforms cannot reproduce physiologically relevant aspects of native tissue such as cell-cell interactions, proliferation, mechanical stimuli, and tissue development. Cancer organoids have been shown to be reliable platforms for modeling tumor growth and drug screening due to their ability to preserve physiologically relevant aspects of native tumor microenvironments. However, their widespread usage to guide clinical decisions is hindered by the time-limited production of organoids and their inherent lack of 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. Unlike traditional droplet emulsion microfluidics that generate micro-organoids with low yield, slow culture time, and limited matrix composition, our pipette droplet technology can be automated using liquid handling instruments and used with both natural and synthetic matrices.


Materials and Methods:: We synthesized norbornene-modified hyaluronic acid (NorHA) polymers, which serve as extracellular matrix (ECM) mimics, to create the cell-encapsulation platform. The droplet generation was carried out with suspended ovarian (OVCAR 5) and prostate (LNCaP) cancer cells (4x10⁶ cells/mL) via water-in-oil (W/O) emulsification. We achieved the rapid generation of droplets via covalent crosslinking by thiol-Nor complexes. RGD motifs were added to the gel to improve cell adhesion, while a combination of DTT and MMP-sensitive motifs was used as crosslinkers to control cell proliferation rate. Mitochondrial activity quantification through WST-1 was performed to study cell proliferation within the microgels. Confocal microscopy was used to assess the volumetric change of the multicellular constructs, and the permeability of the microgels was evaluated through TRITC-labeled dextran (4400 KDa) to determine the drugs' diffusion capacity through the gels. Finally, screening with doxorubicin (DOX) and paclitaxel (PTX) was used to screen the sensitivity of the micro-organoids towards cancer drugs.

Results, Conclusions, and Discussions:: An automated system for pipetting was used to generate microgels and test the effect of pipetting cycles and flow rate on the droplets sizes (Fig. 1A). The results showed that by increasing the pipetting cycles the uniformity of the droplets was improved (Fig. 1B). Moreover, for the cell encapsulation we observed that upon separation, a large quantity of droplets coalesced releasing cells that remained floating freely, which suggested that the system was unoptimized (Fig. 1C). Hence, further experiments were carried out to increase the polymer concentration up to 20 mg/mL, being able to observe a higher microgels separation efficiency (up to 80%), with fewer free-floating cells (Fig. 1D). The permeability of the microgels was evaluated using TRITC-Dextran (4400 KDa), which revealed that fluorophore internalization after 30 minutes, with accumulation and aggregation seen after ~1 hour (Fig. 1E). Finally, the mitochondrial activity assessment exhibited an increase in cell count was ~47% (Fig. 1F), with the relative proliferation of encapsulated cells being slightly lower than in monolayer culture (Fig. 1G).
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.


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