Generation of Three-Dimensional Spheroids/Organoids from Two-Dimensional Cell Cultures Using a Novel Stamp Device

Author(s)

Capistrano-Melo, Henrique ; Silva, Isaura Beatriz Borges ; Gama, Ivan Vieira ; Sogayar, Mari Cleide ; Mantovan, Marluce da Cunha

• Document type: Journal article
• Source: JOVE-JOURNAL OF VISUALIZED EXPERIMENTS; v. N/A, n. 217, p. 12-pg., 2025-03-01.

Abstract

Three-dimensional (3D) cell cultures provide a more accurate representation the in vivo microenvironment than conventional two-dimensional (2D) cultures, since they promote enhanced interactions among cells and the extracellular matrix. This study aimed to develop an efficient, cost-effective, and reproducible methodology to generate 3D cell structures (spheroids/organoids) using an innovative stamp-based system to create microwells in agarose molds.A novel stamp was used to produce 663 microwells per well of a 6-well plate, providing an ideal environment for cell aggregation. Primary porcine pancreatic islet cells were seeded into these microwells, where they aggregated to form spheroids/organoids. The cultures were incubated at 37 degrees C under 5% CO2, and the medium was replaced every 3 days. Spheroid formation was periodically monitored, and samples were collected for characterization. The method successfully generated uniform and high quality spheroids, reducing experimental variability, minimizing manipulation, and enhancing cell interactions. The use of agarose-based micropattern molds provided a simplified, controlled environment for 3D cultures, offering a standardized and costeffective solution.This methodology supports applications for drug testing and tissue engineering, offering a practical and scalable platform for 3D cell culture models that can be easily implemented in various laboratory settings. (AU)

• FAPESP's process: 16/05311-2 - Regenerative medicine aiming at therapy for chronic degenerative diseases (cancer and diabetes)
• Grantee: Mari Cleide Sogayar
• Support Opportunities: Research Projects - Thematic Grants