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Updated: Aug 3, 2026
Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in Microtiter Plates
Published on: March 7, 2017
Evaporation-reducing Culture Condition Increases the Reproducibility of Multicellular Spheroid Formation in
Viswanath Das1, Tomáš Fürst1, Soňa Gurská1
1Institute of Molecular and Translational Medicine, Palacky University in Olomouc.
Abstract:
Tumor models that closely imitate in vivo conditions are becoming increasingly popular in drug discovery and development for the screening of potential anti-cancer drugs. Multicellular tumor spheroids (MCTSes) effectively mimic the physiological conditions of solid tumors, making them excellent in vitro models for lead optimization and target validation. Out of the various techniques available for MCTS culture, the liquid-overlay method on agarose is one of the most inexpensive methods for MCTS generation. However, the reliable transfer of MCTS cultures using liquid-overlay for high-throughput screening may be compromised by a number of limitations, including the coating of microtiter plates (MPs) with agarose and the irreproducibility of uniform MCTS formation across wells. MPs are significantly prone to edge effects that result from excessive evaporation of medium from the exterior of the plate, preventing the use of the entire plate for drug tests. This manuscript provides detailed technical improvements to the liquid-overlay technique to increase the scalability and reproducibility of uniform MCTS formation. Additionally, details on a simple, semi-automatic, and universally applicable software tool for the evaluation of MCTS features after drug treatment is presented.
Insights
This study improves the liquid-overlay method for generating multicellular tumor spheroids (MCTSes), enhancing reproducibility and scalability for anti-cancer drug screening. A new software tool aids in evaluating MCTS features post-treatment.
Area of Science:
- Oncology
- Drug Discovery
- Biotechnology
Background:
- Multicellular tumor spheroids (MCTSes) are valuable in vitro models for mimicking solid tumor physiology in anti-cancer drug discovery.
- The liquid-overlay method using agarose is a cost-effective technique for MCTS generation but faces challenges in scalability and reproducibility for high-throughput screening.
- Limitations include agarose coating issues, inconsistent spheroid formation, and edge effects in microtiter plates (MPs) due to medium evaporation.
Purpose of the Study:
- To present technical improvements to the liquid-overlay method for generating MCTSes.
- To enhance the scalability and reproducibility of uniform MCTS formation for drug screening.
- To introduce a software tool for semi-automatic evaluation of MCTS features after drug treatment.
Main Methods:
- Detailed technical modifications to the standard liquid-overlay MCTS culture technique.
- Implementation of strategies to mitigate edge effects and improve MCTS uniformity.
- Development of a universally applicable, semi-automatic software tool for MCTS analysis.
Main Results:
- Achieved increased scalability and reproducibility in generating uniform multicellular tumor spheroids.
- Successfully addressed limitations associated with agarose coating and edge effects in microtiter plates.
- Demonstrated the utility of the developed software for efficient MCTS feature evaluation post-drug treatment.
Conclusions:
- The refined liquid-overlay technique offers a more robust and scalable approach for MCTS generation in anti-cancer drug development.
- The integrated software tool facilitates standardized and efficient analysis of MCTS responses to drug candidates.
- These advancements improve the reliability of in vitro tumor models for lead optimization and target validation.
