Facile, reproducible, and high-throughput production of human dermal fibroblast spheroids

Spheroids offer a three-dimensional environment that more closely mimics physiologic conditions than traditional two-dimensional cultures. However, conventional spheroid formation methods are often time-consuming, labor-intensive, and struggle to produce high-throughput and uniform-sized spheroids. This work demonstrated an efficient, high-throughput method for generating human dermal fibroblast (HDF) spheroids (up to 1,200 spheroids/well) within 24 h using AggreWell™ plates. Spheroid formation was evaluated over 14 days using two initial seeding densities (100 and 300 cells per microwell). Larger spheroids, ranging from 127–142 µm in diameter, were observed with the higher seeding density, compared to 88–108 µm at the lower seeding density. The spheroids exhibited consistent and uniform size distributions, with a coefficient of variation under 11% on day 1 for both conditions. Key morphological parameters, including perimeter, circularity, spheroid area, and aspect ratio, were assessed on days 1, 7, and 14, revealing stable values, with circularity and aspect ratio remaining above 0.86 and 0.90, respectively. Cell viability exceeded 90% by day 7 and remained above 85% on day 14 for both seeding densities. These results highlight the scalability and reproducibility of HDF spheroid formation for drug screening, tissue engineering, and regenerative medicine.