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Updated: Sep 10, 2025
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A Simple Hanging Drop Cell Culture Protocol for Generation of 3D Spheroids
Published on: May 6, 2011
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SpheroMold: modernizing the hanging drop method for spheroid culture
Ana Paula Pereira Guimaraes1, Italo Rodrigo Calori1,2, Hong Bi3
1Department of Chemistry, Center of Nanotechnology and Tissue Engineering- Photobiology and Photomedicine Research Group, Faculty of Philosophy, Sciences and Letters of Ribeirao Preto, University of Sao Paulo, Sao Paulo, Brazil.
Frontiers in Drug Delivery
|August 21, 2025
Summary
A novel 3D-printed support, SpheroMold, prevents droplet coalescence in hanging drop cultures. This innovation enhances 3D spheroid production per area and simplifies handling for improved cell culture.
Area of Science:
- Biotechnology
- Cell Biology
- Materials Science
Background:
- The hanging drop method is a cost-effective technique for 3D spheroid culture.
- Challenges include droplet coalescence and limited spheroid production per area during Petri dish handling.
Purpose of the Study:
- To introduce SpheroMold, a 3D printing-based support system.
- To enhance spheroid production efficiency and simplify handling in hanging drop cultures.
Main Methods:
- Fabrication of a 3D printing-based negative mold (SpheroMold) using stereolithography.
- Production of the SpheroMold by casting Sylgard® 184 silicone.
- Attachment of the SpheroMold to Petri dish lids for spheroid culture.
Main Results:
- The SpheroMold effectively prevented droplet coalescence during Petri dish inversion.
- Enabled production of numerous 3D spheroids within a limited area.
- Facilitated a larger culture medium volume per drop, potentially reducing medium exchange frequency.
Conclusions:
- SpheroMold is a generalizable method for improving 3D spheroid culture using the hanging drop technique.
- The system enhances spheroid yield, simplifies manipulation, and offers potential for reduced labor intensity.
- This approach advances 3D cell culture techniques for research and biopharmaceutical applications.
