(F-239) Microwells for Uniform Growth of Cancer Spheroids and Organoids

Saturday, October 14, 2023

9:30 AM – 10:30 AM PDT

Location: Exhibit Hall - Row F - Poster # 239

Presenting Author(s)

CS

Cynthia Serrano Marquez (she/her/hers)

Understand Student Research
Lehigh University
Casselberry, Florida, United States

Primary Investigator(s)

YL

Yaling Liu

Professor
Lehigh University, United States

Introduction:: Cancer is a relatively common and dangerous disease, with 1,958,310 new cases and 609,820 new deaths expected in the United States in 2023, it is urgent to research the disease and possible treatments [1]. For decades, animal models have been used for this purpose but are limited by cost, time, and the differences between animal and human pathology and tumorigenesis [2]. An alternative to this animal model is the aggregation of cells into spheroids and organoids because they mimic the cell-cell and cell-matrix interactions found in vivo. Still, traditional methods for this cell aggregation lack reproducibility, are labor-intensive and are time-consuming. We used micropatterned three-dimensional printed stamps to produce microwells in agarose hydrogel on a 24-well plate for the generation of Human Colorectal Carcinoma Spheroids (HCT116) and Colon Cancer Organoids (CCO).

Materials and Methods:: File for the 3D printed stamps was obtained from Professor Gonzalez-Fernandez [3], modified by scaling the tip dimensions by half using SolidWorks, and both files were printed using a FormLabs SLA printer. A 1.5% agarose solution prepared using agarose and distilled water was autoclaved for 30 minutes at 121℃. The stamps were placed in the wells and the solution was pipetted into the wells using the guide channels in the stamp, the stamps were carefully removed after 5 minutes. HCT116 and CCO were placed on separate centrifuge tubes and centrifuged for 5 minutes, at room temperature and at a speed of 2.4 rpm. The supernatant was removed, PBS was added, the tubes were centrifuged with the same settings, and the supernatant was removed. For HCT116, 3mL of Trypsin-EDTA was added and the tube was left in an incubator for 5 minutes. For CCO, 7mL of TrypLE was added and the tube was placed in an orbital shaker at 20 rpm inside an incubator for 15-20 minutes. The same amount of medium was added to the tubes and they were centrifuged again. The supernatant was removed, 1mL of the medium was added, and cells were counted using a Bright-Line Hemacytometer. Approximately 100,000 cells were added to each well and the medium was changed as needed. CCO medium was mixed with 1% matrigel.

Results, Conclusions, and Discussions:: Both stamps can be effectively used to produce Colon Cancer Organoids within a 75µm range within a day from seeding. This range is reduced to approximately 50µm for the 2mm original stamp and 20µm for the 1mm stamps by day 3. For Human Colorectal Carcinoma Spheroids, both stamps produce extremely small spheroids, but the 1mm stamp produces a smaller range by day 3, a significant improvement from the original stamp, as they do not just have a greater range but also a few low outliers. In conclusion, the stamps with lower dimension tips are more consistent in the production of CCO and HCT organoids and spheroids within 3 days from seeding. In addition to a lower range for the length of the organoids/spheroids formed, the updated stamp c a greater number of spheroids than the original stamp, about 100, more than 3 times the original stamp. Although the length of the organoids/spheroids is smaller on the new stamps, it makes the process of producing them less costly and time-consuming, as it uses the same amount of material and time to produce a greater number of organoids/spheroids overall.

Acknowledgements (Optional): :

References (Optional): :

1. Siegel RL, Miller KD, Wagle NS, Jemal A. Cancer statistics, 2023. CA Cancer J Clin. 2023 Jan;73(1):17-48.

2. Kretzschmar K. Cancer research using organoid technology. J Mol Med (Berl). 2021 Apr;99(4):501-515.

3. Gonzalez-Fernandez T, Tenorio AJ, Leach JK. Three-Dimensional Printed Stamps for the Fabrication of Patterned Microwells and High-Throughput Production of Homogeneous Cell Spheroids. 3D Print Addit Manuf. 2020 Jun 1;7(3):139-147.Beghin, A., Grenci, G., Sahni, G. et al. Automated high-speed 3D imaging of organoid cultures with multi-scale phenotypic quantification. Nat Methods 19, 881–892 (2022).