High-throughput 3D spheroid culture and drug testing using a 384 hanging drop array
Open accessAbstract
Culture of cells as three-dimensional (3D) aggregates can enhance in vitro tests for basic biological research as well as for therapeutics development. Such 3D culture models, however, are often more complicated, cumbersome, and expensive than two-dimensional (2D) cultures. This paper describes a 384-well format hanging drop culture plate that makes spheroid formation, culture, and subsequent drug testing on the obtained 3D cellular constructs as straightforward to perform and adapt to existing high-throughput screening (HTS) instruments as conventional 2D cultures. Using this platform, we show that drugs with different modes of action produce distinct responses in the physiological 3D cell spheroids compared to conventional 2D cell monolayers. Specifically, the anticancer drug 5-fluorouracil (5-FU) has higher anti-proliferative effects on 2D cultures whereas the hypoxia activated drug commonly referred to as tirapazamine (TPZ) are more effective against 3D cultures. The multiplexed 3D hanging drop culture and testing plate provides an efficient way to obtain biological insights that are often lost in 2D platforms.
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PDMS wells with triangular pyramid shape opening promoted growth of spheroids, with some control over size achievable.
“PDMS wells with a triangular pyramid shape opening promoted the growth of spheroids, with some control over the size achievable.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [110]
The Experimental section (‘Plate design, fabrication, and hanging drop formation’; page unavailable) says the plate is made of polystyrene, not PDMS, and describes circular access holes and plateaus by diameter, not triangular-pyramid openings. The Results section (‘Formation of hanging drops for spheroid culture’; page unavailable) does support spheroid formation, while the Introduction says the platform forms uniformly sized spheroids. The claimed well material and opening shape are contrary to the paper’s design description. (LLM, conf=0.90)
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Experimental — accuracy not yet measuredIn A431.H9 3D cultures, the lack of proliferating cells resulted in a 100-fold increase in resistance to 5-FU by culture method.
“Tung et al . 64 showed that the lack proliferating cells in A431.H9 3D cultures resulted in 100-fold increase in resistance to 5-FU by culture method.”
From Three-dimensional cell culture systems and their applications in drug discovery and cell-based biosensors (2014) · cites this paper as [64]
The Results and discussion section, under “Anticancer drug sensitivity testing” (page not provided), reports greater 5-FU resistance in A431.H9 spheroids than in 2D cultures. Its IC50 values imply a range of roughly 10- to 1,000-fold, not a single 100-fold increase. It attributes resistance partly to quiescent cells and limited drug penetration, rather than a complete lack of proliferating cells; the same section describes proliferating cells at the spheroid periphery. (LLM, conf=0.88)
Experimental — accuracy not yet measuredTorisawa et al. created a chip system utilizing microfluidics that allows spheroids to grow in a controlled environment for long-term culture.
“Torisawa et al. (110) created a novel chip system utilizing microfluidics that allows spheroids to grow in a controlled environment for long-term culture.”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [110]
The Introduction and the Results and discussion section on long-term culture support that the authors developed a platform for stable, long-term spheroid culture. But the Experimental section describes their system as an injection-molded 384 hanging-drop plate, not a microfluidic chip; the Introduction discusses microfluidic devices as other approaches. Torisawa is a coauthor, although Tung is the paper’s first author. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe device maintained spheroid cultures for up to 2 weeks.
“This device was able to maintain spheroid cultures for up to 2 wk (110).”
From Modeling physiological events in 2D vs. 3D cell culture (2017) · cites this paper as [110]
The Results and discussion section, under “Long-term culture of spheroids in hanging drops” (page unavailable), reports viable spheroids after 12 days of culture. This supports long-term maintenance with the device, but the copy does not establish the claimed two-week (14-day) duration. (LLM, conf=0.85)
Experimental — accuracy not yet measured
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- Authors
- Tung, Y.-C., Hsiao, A. Y., Allen, S. G., Torisawa, Y.-S., Ho, M., & Takayama, S.
- Affiliations
- University of Michigan (US)
- National Institutes of Health (US)
- Corresponding author
- Takayama, Shuichi
- Venue
- The Analyst
- Publisher
- Royal Society of Chemistry (RSC)
- Published
- 2011
- Volume
- 136
- Issue
- 3
- Pages
- 473-478
- ISSN
- 0003-2654 (ISSN-L)
- 1364-5528
- PMC
- PMC7454010
- OpenAlex
- W2045387133
- Cited by
- 969
- References
- 21
- Citations by year
- 2026: 23 · 2025: 46 · 2024: 56 · 2023: 61 · 2022: 77 · 2021: 74 · 2020: 83 · 2019: 64 · 2018: 79 · 2017: 103 · 2016: 81 · 2015: 71 · 2014: 57 · 2013: 54 · 2012: 27
- FWCI
- 17.56
- Type
- journal article
- Language
- en
- Open access
- green
- www.ncbi.nlm.nih.gov
- Locations
- The Analyst (publishedVersion)
- PubMed (publishedVersion)
- PubMed Central (acceptedVersion)
- Scholarworks@UNIST (Ulsan National Institute of Science and Technology) (submittedVersion)
- Funders
- National Institutes of Health
- National Cancer Institute (Z01BC010891)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Physical Sciences › Engineering › Electrical and Electronic Engineering › 3D IC and TSV technologies
- Keywords
- multicellular spheroids, hanging drop culture, 384-well plate, high-throughput screening, drug testing, 5-fluorouracil, tirapazamine, anticancer agents, antiproliferative activity
- MeSH terms
- Antineoplastic Agents, Cell Survival, Drug Evaluation, Preclinical, Humans, Osmolar Concentration, Time Factors, Spheroids, Cellular, Cell Culture Techniques, Cell Line, Tumor, High-Throughput Screening Assays
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