Related Experiment Video
Updated: Apr 23, 2026
Generation of High-Throughput Three-Dimensional Tumor Spheroids for Drug Screening
Published on: September 5, 2018
Optimization of Aqueous Biphasic Tumor Spheroid Microtechnology for Anti-Cancer Drug Testing in 3D Culture
Stephanie Lemmo1, Ehsan Atefi1, Gary D Luker2
1Department of Biomedical Engineering, The University of Akron, Akron, Ohio 44325, United States.
Abstract:
Tumor spheroids are three-dimensional clusters of cancer cells that exhibit characteristics of poorly perfused tumors and hence present a relevant model for testing the efficacy of anti-cancer compounds. The use of spheroids for drug screening is hindered by technological complexities for high throughput generation of consistent size spheroids individually addressable by drug compounds. Here we present and optimize a simple spheroid technology based on the use of an aqueous two-phase system. Cancer cells confined in a drop of the denser aqueous dextran phase are robotically dispensed into a microwell containing the immersion aqueous polyethylene glycol phase. Cells remain within the drop and form a viable spheroid, without a need for any external stimuli. The size of resulting spheroids is sensitive to volume variations of dispensed drops from the air displacement pipetting head of a commercial liquid handling robot. Therefore, we parametrically optimize the process of dispensing of dextran phase drops. For a given cell density, this optimization reproducibly generates consistent size spheroids in standard 96-well plates. In addition, we evaluate the use of a commercial biochemical assay to examine cellular viability of cancer cell spheroids. Spheroids show a dose-dependent response to cisplatin similar to a monolayer culture. However unlike their two-dimensional counterpart, spheroids exhibit resistance to paclitaxel treatment. This technology, which uses only commercially-available reagents and equipment, can potentially expedite anti-cancer drug discovery. Although the use of robotics makes the ATPS spheroid technology particularly useful for drug screening applications, this approach is compatible with simpler liquid handling techniques such as manual micropipetting and offers a straightforward method of 3D cell culture in research laboratories.
Insights
We developed a simple, reproducible method using an aqueous two-phase system (ATPS) to create consistent 3D tumor spheroids for anti-cancer drug screening. This technology aids in discovering new cancer therapies.
Area of Science:
- Biotechnology
- Cancer Research
- Drug Discovery
Background:
- Tumor spheroids are valuable 3D cancer models for drug screening due to their resemblance to poorly perfused tumors.
- Current spheroid generation methods face technological challenges in high-throughput, consistent production.
- Addressing these limitations is crucial for advancing anti-cancer compound efficacy testing.
Purpose of the Study:
- To present and optimize a novel, simple spheroid technology utilizing an aqueous two-phase system (ATPS).
- To establish a reproducible method for generating consistently sized, individually addressable tumor spheroids.
- To evaluate the utility of this ATPS spheroid technology for anti-cancer drug screening and 3D cell culture.
Main Methods:
- A simple spheroid technology was developed using an aqueous two-phase system (ATPS) with dextran and polyethylene glycol (PEG) phases.
- Cancer cells in dextran drops were robotically dispensed into PEG microwells, forming viable spheroids without external stimuli.
- Parametric optimization of drop dispensing was performed to ensure consistent spheroid size, sensitive to volume variations.
Main Results:
- The optimized ATPS method reproducibly generated consistent-sized tumor spheroids in 96-well plates for a given cell density.
- Cellular viability assays showed spheroids responded dose-dependently to cisplatin, similar to 2D monolayer cultures.
- Tumor spheroids demonstrated resistance to paclitaxel, unlike their 2D counterparts, highlighting differences in 3D drug response.
Conclusions:
- The ATPS spheroid technology offers a straightforward, scalable method for 3D cell culture and anti-cancer drug discovery.
- This technology uses commercially available reagents and equipment, compatible with both robotic and manual liquid handling.
- The ability to generate consistent spheroids and reveal differential drug responses can expedite the identification of effective anti-cancer compounds.
