Optimization of liquid overlay technique to formulate heterogenic 3D co-cultures models
Abstract
Three-dimensional (3D) cell culture models of solid tumors are currently having a tremendous impact in the in vitro screening of candidate anti-tumoral therapies. These 3D models provide more reliable results than those provided by standard 2D in vitro cell cultures. However, 3D manufacturing techniques need to be further optimized in order to increase the robustness of these models and provide data that can be properly correlated with the in vivo situation. Therefore, in the present study the parameters used for producing multicellular tumor spheroids (MCTS) by liquid overlay technique (LOT) were optimized in order to produce heterogeneous cellular agglomerates comprised of cancer cells and stromal cells, during long periods. Spheroids were produced under highly controlled conditions, namely: (i) agarose coatings; (ii) horizontal stirring, and (iii) a known initial cell number. The simultaneous optimization of these parameters promoted the assembly of 3D characteristic cellular organization similar to that found in the in vivo solid tumors. Such improvements in the LOT technique promoted the assembly of highly reproducible, individual 3D spheroids, with a low cost of production and that can be used for future in vitro drug screening assays.
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Multicellular tumor spheroids provide a platform that more accurately mimics the architecture, diffusion constraints, and cellular interactions present in solid tumors.
“Among emerging in vitro models, multicellular tumor spheroids provide a platform that more accurately mimics the architecture, diffusion constraints, and cellular interactions present in solid tumors [112,182,183].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [182]
Incorporating stromal cell types makes spheroid models more closely replicate the cellular composition of in vivo tumors.
“By incorporating stromal cell types, such as fibroblasts, endothelial cells, or immune cells, these models more closely replicate the cellular composition of in vivo tumors [182].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [182]
The plateau phase reflects the onset of metabolic stress and reduced proliferative capacity, closely mirroring the dormancy observed in early-stage solid tumors.
“This plateau reflects the onset of metabolic stress and reduced proliferative capacity, closely mirroring the dormancy observed in early-stage solid tumors [48,182,221].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [182]
A range of microscopic techniques has been employed to evaluate spheroid growth, morphology, and internal cellular organization.
“A range of microscopic techniques has been employed to evaluate spheroid growth, morphology, and internal cellular organization [14,182,232].”
From Modeling Tumor Microenvironment Complexity In Vitro: Spheroids as Physiologically Relevant Tumor Models and Strategies for Their Analysis (2025) · cites this paper as [182]
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References 57 · Cited by 156
- Authors
- Costa, E. C., Gaspar, V. M., Coutinho, P., & Correia, I. J.
- Affiliations
- University of Beira Interior (PT)
- Corresponding author
- Correia, Ilídio J.
- Venue
- Biotechnology and Bioengineering
- Publisher
- Wiley
- Published
- 2014-02-25
- Volume
- 111
- Issue
- 8
- Pages
- 1672-1685
- ISSN
- 0006-3592 (ISSN-L)
- 1097-0290
- OpenAlex
- W2100432154
- Cited by
- 156
- References
- 57
- Citations by year
- 2026: 9 · 2025: 13 · 2024: 14 · 2023: 13 · 2022: 10 · 2021: 20 · 2020: 18 · 2019: 15 · 2018: 17 · 2017: 13 · 2016: 8 · 2015: 3 · 2014: 2
- FWCI
- 4.50
- Type
- journal article
- Language
- en
- Open access
- closed
- Locations
- Biotechnology and Bioengineering (publishedVersion)
- PubMed (publishedVersion)
- Portuguese National Funding Agency for Science, Research and Technology (RCAAP Project by FCT) (submittedVersion)
- Topics
- Physical Sciences › Engineering › Biomedical Engineering › 3D Printing in Biomedical Research
- Health Sciences › Medicine › Oncology › Cancer Cells and Metastasis
- Physical Sciences › Mathematics › Modeling and Simulation › Mathematical Biology Tumor Growth
- Keywords
- tumor spheroids, 3D cell culture models, tumor stroma, cancer cells, in vitro drug testing, tumor heterogeneity
- MeSH terms
- Drug Screening Assays, Antitumor, Equipment Design, Humans, Tumor Cells, Cultured, Spheroids, Cellular, Coculture Techniques, Cell Line, Tumor
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