Poster H6 - Micro-Organoid Generation by Gel-Droplet Platform for Cancer Drug Screening

Friday, October 25, 2024

3:30 PM – 4:30 PM EST

Location: Exhibit Hall E, F & G

Presenting Author(s)

AT

Angela Taglione

Undergraduate Student
University of Notre Dame, United States

Co-Author(s)

Daniel Montes Pinzon

PhD Student
University of Notre dame
South Bend, Indiana, United States
Fei Fan, PhD (he/him/his)

Postdoc Researcher
Michigan State University
East Lansing, Michigan, United States
LC

Liao Chen

Research Scientist at Elkem Silicones
Elkem Silicones, United States
XL

Xin Lu

John M. and Mary Jo Boler Collegiate Associate Professor
University of Notre Dame, United States
SS

Sharon Stack

Professor and Director, Harper Cancer Research Institute, University of Notre Dame
Harper Cancer Research Institute, United States

Primary Investigator(s)

Hsueh-Chia Chang, Phd

Bayer Professor of Chemical and Biomolecular Engineering
University of Notre Dame
Notre Dame, Indiana, United States

Last Author(s)

Donny Hanjaya-Putra

Assistant Professor
University of Notre Dame
Notre Dame, Indiana, United States

Introduction: Personalized medicine is an important topic in contemporary healthcare, aiming to tailor medical treatment to individual patients. However, 2D modeling platforms are not as physiologically relevant as 3D models because they cannot reproduce relevant aspects of native tissue such as cell-cell interactions, proliferation, the extracellular matrix (ECM), and tissue development. Spheroids are a type of 3D model that offer a superior representation of solid tumor complexities compared to other models. Despite their advantages, spheroid production is limited due to time consumption and reproducibility. Given that treatment decisions are often required within 14 days of diagnosis, there is a pressing need for a more efficient fabrication method to ensure timely and effective translation to clinical settings. To address these challenges, we evaluated the production of microgels using an elliptical pipette for cell encapsulation by droplet emulsion, for high-throughput drug screening. This pipette droplet technology offers significant improvements over traditional droplet emulsion microfluidics, which typically yield micro-organoids at a low rate, require extended culture times, and restrict matrix composition choices. In addition, this method supports automation via liquid handling instruments and is adaptable to both natural and synthetic matrices.

Materials and

Methods: Norbornene-modified hyaluronic acid (NorHA) polymers were synthesized to mimic the ECM and create the cell-encapsulation platform. Spheroids and microgels were fabricated using the NorHA solution, which included crosslinkers DTT, MMP-sensitive motifs, and RGD for cell adhesion, prepared with Irgacure “I2959” as a photoinitiator. Cell encapsulation was performed via water-in-oil emulsion using a custom elliptical pipette tip. The droplets were polymerized under UV light to form microgels, then cultured in optimized conditions for cell proliferation and drug screening. Cell cultures of ovarian (RFP-OVCAR5) and prostate (LNCaP) cancer lines were maintained under standard conditions and used for experiments at a concentration of 4x10^6 cells/mL. Proliferation and viability assays were performed using mitochondrial activity quantification (WST-1) and confocal microscopy to assess cell growth. For anti-cancer drug screening, encapsulated spheroids were exposed to varying concentrations of doxorubicin and enzalutamide, with IC50 values determined by evaluating cell viability 24 hours post-treatment.

Results, Conclusions, and Discussions: An automated pipetting system was used to generate microgels and test the effect of pipetting cycles and flow rate on droplet sizes (Fig. 1AB). Increasing the flow rate did not affect droplet uniformity, while increasing the aspiration/dispensing cycles greatly improved it (Fig. 1C). Mitochondrial activity assessment showed a ~47% increase in cell count, with encapsulated cell proliferation slightly lower than in monolayer culture (Fig. 2). The platform used a combination of MMP-sensitive and DTT crosslinkers. MMP-sensitive crosslinker, being degradable, imposed less constraint on the cells, allowing higher proliferation rates as cells modified their ECM, promoting tissue growth (Fig. 3).

Viability assays assessed cell health and survival within the gel platform before drug screening. OVCAR5 and LNCaP had 76.7% and 77.3% viability, respectively, after 24 hours of encapsulation (Fig. 4). After 8 days, LNCaP viability increased to 90.98%, due to cell proliferation within the microgels platform. The IC50 was estimated from preliminary cancer drug screening using DOX and ENZ. The IC50 values for LNCaP treated with DOX were 0.037 μM, with ENZ at 1 μM, and for OVCAR5 treated with DOX at 0.3 μM (Fig. 5).

Acknowledgements (Optional): Special thanks to the members of the Hanjaya-Putra and Chang labs, and especially to graduate student Daniel Montes Pinzon for his guidance and mentorship throughout this project.