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    Tissue Engineering

    (K-430) Development of a Mechanically Tuned In Vitro Ovarian Cancer Organoid Model

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row K - Poster # 430

      Presenting Author(s)

    • Astrid Daugherty (she/her/hers)

      Research Associate
      FAMU-FSU College of Engineering
      Tallahassee, Florida, United States

      • Co-Author(s)

    • MS

      Mary Jean Savitsky

      PhD Student
      FAMU-FSU College of Engineering, United States

    • Izabela Kowalik, UG

      Research Associate
      National High Magnetic Field Lab
      Land O' Lakes, Florida, United States

    • MA

      Makena Andersen

      Research Associate
      Lewis & Clark College, Department of Chemistry & Physics
      Tallahassee, Florida, United States

    • AM

      Amisha Martin

      Research Associate
      FAMU-FSU College of Engineering, United States

    • NK

      Navneet Kaur

      Postdoctoral Fellow
      FAMU-FSU College of Engineering, United States

      • Last Author(s)

    • JA

      Jamel Ali

      Professor
      FAMU-FSU College of Engineering, Florida, United States

    Introduction::
    Effective screening and treatment of ovarian cancer is a major challenge in the Western world with high mortality and relapse rates due to delayed detection and limited treatment. Three dimensional (3D) ovarian organoid models offer the potential for better understanding the disease and developing effective therapies. However, current models largely neglect the mechanical properties of native ovarian carcinoma and the surrounding extracellular matrix (ECM) which can greatly influence cell behavior in vivo. This investigation explores how altering the stiffness, of a rheologically characterized artificial ECM hydrogel surrounding ovarian cancer cells, influences cell viability, growth, and structural development within a 3D ovarian cancer tissue model.




    Materials and Methods:: An epithelial ovarian cancer cell line (SKOV3, ATCC HTB-77) was cultured and expanded following American Type Culture Collection guidelines. Artificial ECM hydrogels consisted of 4 wt./vol.% gelatin (type A, porcine skin, MilliporeSigma) with varying alginate (medium viscosity, 773kDa, MilliporeSigma). concentrations (3, 4, and 5 wt./vol.%). Rheological analysis was preformed using an Anton Paar rheometer with a 25 mm diameter parallel plate geometry. Measurements were obtained at 37 ºC before with a geometry gap of 1 mm, and included flow curves, strain sweeps, and frequency sweeps. Scanning electron microscopy (SEM) illustrated structural differences between glutaraldehyde-fixed gels with and without cancer cells. Live/dead assays using Calcein-AM and ethidium homodimer-1 were performed on 2D and 3D cultures. MTT assays were performed to assess proliferation rates.


    Results, Conclusions, and Discussions:: Results: 2D culture of SKOV3 cell lines were observed to have typical cell growth, reaching ~30% - 40% confluency within 24 hours, as observed via phase-contrast microscopy (Fig. 1A). Rheological analysis of alginate-gelatin hydrogels confirmed the presence of viscoelastic properties for the 3D cell culture of organoids, exhibiting shear-thinning behavior and storage moduli values within the natural range of ovarian cancer cells (0.1-10 kPa), as shown in flow curves and shear sweeps (Fig1. B,C). Cell viability assays are expected to reveal a trend linking hydrogel stiffness to the live to dead ratio of seeded cancer cells in the 3D microenvironment. Proliferation assays will show how cell viability varies with matrix stiffness over time. SEM images illustrate the surface characteristics of the material with and without cells, revealing the impact of organoid growth on its microstructure.











    Figure 1. (A) Phase Contrast Image of SKOV3 cells (B) Flow Curve of a 3% wt./vol. alginate, 4% wt. gelatin hydrogel (C) Stress sweep of hydrogel






    Discussion: 2D cell culture and expansion is crucial for initial seeding of cells in 3D culture environments. 2D cultures of SKOV3 cells displayed epithelial morphology and doubling time (48 hrs.). Rheological analysis of the softest hydrogel (3% alginate and 4% gelatin) confirms storage moduli values of ~1 kPa, falling within the target stiffness range for ovarian cancer. Shear-thinning behavior, observed through flow curves (Fig.1B), shows all gels are shear thinning. This is essential for 3D cell culture as it replicates the native mechanical microenvironment ovarian cancer cells develop in. Further experiments are planned to continue the investigation to explore the effects of stromal cell loading on 3D organoid development.





    Conclusions: It was observed that all alginate-gelatin hydrogels display both shear thinning and viscoelastic properties, similar to those previously reported for natural ovarian cancer tissue, enabling more in vivo like cell growth, movement, and function. The ovarian SKOV3 cell line was successfully incorporated into hydrogels and were viable after incorporation. This work will enable future investigations aimed at high-throughput in vitro screening of ovarian cancer interventions that more closely match in vivo physiological outcomes.






    Acknowledgements (Optional): : This work was funded by The Grainger Foundation Frontiers of Engineering Grant under the National Academy of Sciences Award Number: 2000013181, the NSF (EES-1735968, EES-2219558), and DOW. Support was also provided by the National Institute of General Medical Sciences of the National Institutes of Health under Award Number R16GM145595. All work was performed at the National High Magnetic Field Laboratory, which is supported by NSF Cooperative Agreement DMR-1644779 and the State of Florida.


    References (Optional): :