Method for Large-scale Production of hIPSC Spheroids

Area of Science:

• Biotechnology
• Stem Cell Biology
• Tissue Engineering

Background:

• Stem cell spheroids are crucial for tissue engineering, 3D cell models, and fundamental biology.
• Current spheroid generation methods lack efficiency, scalability, and accessibility for large-scale production.
• A need exists for improved, reproducible spheroid production techniques compatible with standard laboratory equipment.

Purpose of the Study:

• To present a novel, accessible, and efficient method for generating large quantities of stem cell spheroids.
• To provide a detailed protocol for producing spheroids using 3D-printed molds and pellet culture.
• To demonstrate the method's effectiveness with human induced pluripotent stem cells (hiPSCs).

Main Methods:

• Utilized 3D printing to create silicone molds with U-shaped microstructures for pellet culture.
• Developed a detailed, illustrated manufacturing protocol for the spheroid induction device.
• Employed the device in standard 6-well plates for spheroid generation from hiPSCs.

Main Results:

• Achieved rapid production of reproducible and controlled hiPSC spheroids (130 ± 10 μm) within 24 hours.
• Enabled the generation of large quantities (2 × 10^4 spheroids per 1 × 10^6 cells) using a reusable, cost-effective mold.
• Demonstrated successful spheroid induction across multiple hiPSC lines with tunable diameters.

Conclusions:

• The novel pellet culture method using 3D-printed molds offers an accessible, efficient, and reproducible way to produce stem cell spheroids.
• This technique is suitable for routine laboratory use, facilitating large-scale spheroid generation for various biotechnological applications.
• Freely available protocols and mold files promote widespread adoption and advancement in stem cell research.