Introduction: It’s estimated that there will be over two million new cancer cases and over six thousand cancer-related deaths in the United States this year [1]. Making it urgent to research the disease and possible treatments. Animal models have been used for decades 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. However, traditional methods for this cell aggregation like the gentle rotational stirring of suspensions and the hanging drop method lack reproducibility, are labor-intensive, and are time-consuming [3]. To address this, we used different micropatterned three-dimensional printed stamps to produce microwells in agarose hydrogel on a 24-well plate, seeded Human Colorectal Carcinoma Spheroids (HCT116) at different densities, to identify the best seeding conditions, treatment, and microwell pattern.
Materials and
Methods: Files for the micropatterned stamps were made using SolidWorks and printed on the Elegoo Saturn 2 3D printer. For control, we used a 2mm tip following the dimensions in reference 3. We reduced the size of the tip to have more spheroids per well with the same amount of materials and time. These reduced-size tips have a 1mm base and two different depths to test which one is more optimal. Additionally, there was also a flat bottom microwell. A 1.5% agarose solution was prepared using agarose and distilled water and 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 side of the stamp, the stamps were carefully removed after 5 minutes. Approximately 100,000 cells were added to low-density wells and 400,000 to high-density wells. The medium was changed as needed. Some wells were treated with 200µl of anti-adhesive rising solution for an hour and watched with PBS before seeding. Some wells had 10% matrigel in the first medium to promote spheroid formation.
Results, Conclusions, and Discussions: Without matrigel and anti-adhesion rising solution spheroids do not properly form, rather the cells just aggregate in a cluster regardless of the seeding cell density. For those wells treated with the anti-adhesion solution and that had matrigel on the first medium, the spheroids started to form in 3 days, only on the depth pyramidal shape tips. Spheroids did not start to form either in the flat bottom microwells or the shallow ones regardless of whether the treatment was within 5 days from seeding. We concluded that deeper and sharper microwells are better for the formation of HCT116 spheroids in short periods at a lower seeding density with matrigel to promote aggregation and formation as well as anti-adhesion solution treatment of the agar before seeding.
