Related Experiment Video
Updated: Mar 25, 2026
Generation of 3D Tumor Spheroids for Drug Evaluation Studies
Published on: February 24, 2023
Comparative analysis of tumor spheroid generation techniques for differential in vitro drug toxicity
Shreya Raghavan1, Pooja Mehta1, Eric N Horst2
1Department of Materials Science and Engineering, University of Michigan, Ann Arbor, USA.
Abstract:
Multicellular tumor spheroids are powerful in vitro models to perform preclinical chemosensitivity assays. We compare different methodologies to generate tumor spheroids in terms of resultant spheroid morphology, cellular arrangement and chemosensitivity. We used two cancer cell lines (MCF7 and OVCAR8) to generate spheroids using i) hanging drop array plates; ii) liquid overlay on ultra-low attachment plates; iii) liquid overlay on ultra-low attachment plates with rotating mixing (nutator plates). Analysis of spheroid morphometry indicated that cellular compaction was increased in spheroids generated on nutator and hanging drop array plates. Collagen staining also indicated higher compaction and remodeling in tumor spheroids on nutator and hanging drop arrays compared to conventional liquid overlay. Consequently, spheroids generated on nutator or hanging drop plates had increased chemoresistance to cisplatin treatment (20-60% viability) compared to spheroids on ultra low attachment plates (10-20% viability). Lastly, we used a mathematical model to demonstrate minimal changes in oxygen and cisplatin diffusion within experimentally generated spheroids. Our results demonstrate that in vitro methods of tumor spheroid generation result in varied cellular arrangement and chemosensitivity.
Insights
Different methods for creating multicellular tumor spheroids impact their structure and drug resistance. Hanging drop and nutator plates yield more compact spheroids with higher chemoresistance compared to standard liquid overlay methods.
Area of Science:
- Oncology
- Biotechnology
- Drug Discovery
Background:
- Multicellular tumor spheroids are valuable in vitro models for preclinical chemosensitivity testing.
- Standard methods for spheroid generation may not fully recapitulate in vivo tumor microenvironments.
Purpose of the Study:
- To compare different spheroid generation techniques for their impact on morphology, cellular arrangement, and chemosensitivity.
- To evaluate the influence of spheroid generation methods on drug response in cancer cell lines.
Main Methods:
- Generated multicellular tumor spheroids using three methods: hanging drop arrays, liquid overlay on ultra-low attachment plates, and liquid overlay with rotation (nutator plates).
- Analyzed spheroid morphometry, collagen content, and cellular compaction.
- Assessed chemosensitivity to cisplatin in generated spheroids.
- Utilized a mathematical model to evaluate oxygen and cisplatin diffusion.
Main Results:
- Spheroids generated via hanging drop and nutator plates exhibited increased cellular compaction and collagen remodeling compared to standard liquid overlay.
- These more compact spheroids demonstrated significantly higher chemoresistance to cisplatin.
- Mathematical modeling indicated minimal diffusion changes across experimental spheroid types.
Conclusions:
- The method of in vitro tumor spheroid generation significantly influences spheroid structure and preclinical chemosensitivity assay outcomes.
- Hanging drop and nutator plate methods produce more chemoresistant spheroids, potentially offering a more predictive preclinical model.
- Careful selection of spheroid generation techniques is crucial for accurate chemosensitivity assessments.
