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    Women's Health

    (M-492) Automating a uterine fibroid organoid culturing system: A preliminary study with MCF7 spheroids

    Thursday, October 12, 2023
    9:30 AM – 10:30 AM PDT
    Location: Exhibit Hall - Row M - Poster # 492
    Introduction:: Uterine fibroids are benign tumors in the myometrium and manifest in over 70% of premenopausal women before the age of fifty.1 While uterine fibroids are benign, they result in high morbidity due to significant gynecologic complications in symptomatic individuals like abnormal uterine bleeding, severe pelvic pain/pressure, and frequent urination.1,2 In the US alone fibroids result in billions of dollars' worth of healthcare costs annually. Their overall impact is likely underestimated due to symptoms that coincide with other pathologies, which also complicates treatment1,2,3,4 Understanding fibroid pathophysiology requires the development of effective research models. Organoids are three-dimensional models that better represent tumor morphogenesis and hormone response than cell monolayers.5 Recently, Banerjee S. et. al cultured uterine fibroid organoids from isolated stem cells.6 We will validate their procedure by isolating stem cells from myometrial and fibroid tissue using the established surface biomarkers Stro-1 and CD44.7 Manually culturing organoids can be labor intensive with potential for human error, so automating our organoid culturing should minimize error and increase throughput.  As a proof of concept, we performed a preliminary study with MCF7 cells, a breast cancer cell line, to automate the culturing of spheroids.8 Successfully culturing spheroids will allow us to identify potential pitfalls for manually culturing fibroid organoids using isolated stem cells and translating our workflow to automation.

    Materials and Methods:: In this proof-of-concept study, we created a spheroid culturing protocol with MCF7 cells. We cultured MCF7 cells in T-75 flasks in 2D culture for 3-7 days until cells were confluent (Fig 1). The media was Dulbecco's Modified Eagle Medium High Glucose, 10% Fetal Bovine Serum, and 1X Antibiotic-Antimycotic. 96-well U-bottom plates were seeded with passaged cells with cell counts ranging from 1000-6000 in 1000 cell increments with 200 µl of media. They were cultured for 5 days, with a media change on day 2. Spheroids were fixed with 20 µl of 4% paraformaldehyde for 30 minutes, permeabilized with 30 µl of 0.1% Triton X-100 for 30 minutes, blocked with 30 µl of 3% BSA blocking solution for 30 minutes, and stained with 30 µl of a 1:1 DAPI and Rhodamine Phalloidin for 30 minutes (Fig 1). After each step, we performed three 100 ul washes with 1X phosphate-buffered saline (PBS) and centrifuged the spheroids for 5 minutes at 1000 rpm to the bottom of the well. Spheroids were imaged on an Olympus FV3000 for confocal imaging and raw images were handled using the FIJI image processing software.9 Using a custom script, we streamlined image processing steps including background correction, cropping, scale bars, and saving. The script calculates the spheroid’s aspect ratio (AR) and circularity (Circ).

    Results, Conclusions, and Discussions::

    MCF7 spheroids were characterized using AR and Circ, numerical calculations that can characterize sphericality. AR is the ratio between the length and depth of z-slices. Circ measures how similar the 2D z projection is to a circle. In Figure 2, z projections of spheroids with different cell counts stained with DAPI and rhodamine phalloidin are shown. Figures 2A-F show a wide range of AR (1.10 - 1.43) with no predictable dependence on their cell count. Despite the spheroids in Figures 2A and 2B having lower cell counts (1000 and 2000), their aspect ratios (1.36 and 1.43) were larger than some spheroids with larger counts. Figures 2A-F also show that circularity had no clear trend. The circularities closest to 1 were spheroids seeded with 1000 and 6000 cells in Figures 2A and 2F. 


    Figures 2B and 2G have fibers (dyed in blue) within the spheroids that are contaminants introduced during manual cell culturing. They can affect the formation of spheroids which can cause irregular conformations. For example, in Figure 2B, the fiber affected the spheroid’s shape by causing MCF7 cells to aggregate around it. The fiber in Figure 2G could be responsible for the large AR difference compared to Figure 2C, 1.23 and 2.76 respectively, despite both being seeded with 3000 cells. Another issue was that manual pipetting occasionally resulted in spheroid loss during plate staining due to spheroids floating in the wells. Incorporating intermediate centrifuge steps resolved this issue.


    Preliminary results suggest that seeding cell density may not impact spheroid shape as much as irregularities and contamination from manual workflows that can introduce extraneous variables and impact spheroid development. The next steps will be to automate spheroid culturing to minimize contamination and spheroid loss, decrease variability within and between cell seeding counts, and increase overall throughput. We will compare these spheroids to manually cultured plates using a workflow revised based on our results. This preliminary work will inform our future manual and automated uterine fibroid culturing. With the future development of uterine fibroid organoids, researchers can draw more comprehensive biological conclusions about their pathophysiology.



    Acknowledgements (Optional): : Special thanks to the Klapperich Lab at Boston University for their support of this project.

    References (Optional): :

    1.        Giuliani E, As-Sanie S, Marsh EE. Epidemiology and management of uterine fibroids. Int J Gynecol Obstet. 2020;149(1):3-9. doi:10.1002/ijgo.13102


    2.        Yang Q, Ciebiera M, Bariani MV, et al. Comprehensive Review of Uterine Fibroids: Developmental Origin, Pathogenesis, and Treatment. Endocr Rev. 2022;43(4):678-719. doi:10.1210/endrev/bnab039


    3.        Cardozo ER, Clark AD, Banks NK, Henne MB, Stegmann BJ, Segars JH. The estimated annual cost of uterine leiomyomata in the United States. Am J Obstet Gynecol. 2012;206(3):211.e1-211.e9. doi:10.1016/j.ajog.2011.12.002


    4.        Aninye IO, Laitner MH. Uterine Fibroids: Assessing Unmet Needs from Bench to Bedside. J Womens Health. 2021;30(8):1060-1067. doi:10.1089/jwh.2021.0280


    5.        Clevers H. Modeling Development and Disease with Organoids. Cell. 2016;165(7):1586-1597. doi:10.1016/j.cell.2016.05.082


    6.        Banerjee S, Xu W, Chowdhury I, et al. Human Myometrial and Uterine Fibroid Stem Cell-Derived Organoids for Intervening the Pathophysiology of Uterine Fibroid. Reprod Sci. 2022;29(9):2607-2619. doi:10.1007/s43032-022-00960-9


    7.        Mas A, Nair S, Laknaur A, Simón C, Diamond MP, Al-Hendy A. Stro-1/CD44 as putative human myometrial and fibroid stem cell markers. Fertil Steril. 2015;104(1):225-234.e3. doi:10.1016/j.fertnstert.2015.04.021


    8.        Froehlich K, Haeger JD, Heger J, et al. Generation of Multicellular Breast Cancer Tumor Spheroids: Comparison of Different Protocols. J Mammary Gland Biol Neoplasia. 2016;21(3):89-98. doi:10.1007/s10911-016-9359-2


    9.        Schindelin J, Arganda-Carreras I, Frise E, et al. Fiji: an open-source platform for biological-image analysis. Nat Methods. 2012;9(7):676-682. doi:10.1038/nmeth.2019