Optimized delivery of siRNA into 3D tumor spheroid cultures in situ
Open accessAbstract
3D tissue culture provides a physiologically relevant and genetically tractable system for studying normal and malignant human tissues. Despite this, gene-silencing studies using siRNA has proved difficult. In this study, we have identified a cause for why traditional siRNA transfection techniques are ineffective in eliciting gene silencing in situ within 3D cultures and proposed a simple method for significantly enhancing siRNA entry into spheroids/organoids. In 2D cell culture, the efficiency of gene silencing is significantly reduced when siRNA complexes are prepared in the presence of serum. Surprisingly, in both 3D tumour spheroids and primary murine organoids, the presence of serum during siRNA preparation rapidly promotes entry and internalization of Cy3-labelled siRNA in under 2 hours. Conversely, siRNA prepared in traditional low-serum transfection media fails to gain matrigel or spheroid/organoid entry. Direct measurement of CTNNB1 mRNA (encoding β-catenin) from transfected tumour spheroids confirmed a transient but significant knockdown of β-catenin when siRNA:liposome complexes were formed with serum, but not when prepared in the presence of reduced-serum media (Opti-MEM). Our studies suggest a simple modification to standard lipid-based transfection protocols facilitates rapid siRNA entry and transient gene repression, providing a platform for researchers to improve siRNA efficiency in established 3D cultures.
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A likely reason for the limited use of siRNA technology in 3D organoid cultures is the technical challenge of delivering siRNAs through the extracellular matrix to organoids in situ.
“The use of siRNA technology in 3D organoid cultures has been very limited [48,49,50], likely also due to the technical challenge of delivering siRNAs through the extracellular matrix to organoids in situ [49].”
From Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers (2024) · cites this paper as [49]
The use of siRNA technology in 3D organoid cultures has been very limited.
“The use of siRNA technology in 3D organoid cultures has been very limited [48,49,50], likely also due to the technical challenge of delivering siRNAs through the extracellular matrix to organoids in situ [49].”
From Regulation of Enterocyte Brush Border Membrane Primary Na-Absorptive Transporters in Human Intestinal Organoid-Derived Monolayers (2024) · cites this paper as [49]
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- Authors
- Morgan, R. G., Chambers, A. C., Legge, D. N., Coles, S. J., Greenhough, A., & Williams, A. C.
- Affiliations
- University of Sussex (GB)
- University of Bristol (GB)
- University of Worcester (GB)
- Corresponding author
- Morgan, R. G.
- Greenhough, A.
- Venue
- Scientific Reports
- Published
- 2018-05-21
- Volume
- 8
- Issue
- 1
- Pages
- 7952-7952
- ISSN
- 2045-2322 (ISSN-L)
- PMC
- PMC5962539
- OpenAlex
- W2800793366
- Cited by
- 47
- References
- 27
- Citations by year
- 2026: 6 · 2025: 12 · 2024: 6 · 2023: 9 · 2022: 4 · 2021: 4 · 2020: 5 · 2019: 1
- FWCI
- 0.89
- Type
- journal article
- Language
- en
- Open access
- gold
- doi.org
- Locations
- Scientific Reports (publishedVersion, cc-by)
- Europe PMC (PubMed Central) (publishedVersion, other-oa)
- PubMed Central (publishedVersion, cc-by)
- PubMed (publishedVersion)
- UWE Research Repository (UWE Bristol) (submittedVersion, cc-by)
- Sussex Research Online (University of Sussex) (submittedVersion, cc-by)
- Worcester Research and Publications (University of Worcester) (submittedVersion)
- DOAJ (DOAJ: Directory of Open Access Journals) (submittedVersion)
- Figshare (submittedVersion, cc-by)
- Explore Bristol Research (submittedVersion, cc-by)
- Bristol Research (University of Bristol) (submittedVersion, cc-by)
- Funders
- Cancer Research UK (C19/A11975;, A11975)
- University of Bristol
- Medical Research Council (MR/N001494/1)
- Beatson Institute for Cancer Research
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA Interference and Gene Delivery
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Molecular Biology Techniques and Applications
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › RNA and protein synthesis mechanisms
- Keywords
- siRNA delivery, tumor spheroids, organoids, gene silencing, CTNNB1, Matrigel
- MeSH terms
- Animals, Humans, Organoids, Tumor Cells, Cultured, Colorectal Neoplasms, Gene Transfer Techniques, Spheroids, Cellular, Cell Culture Techniques, Gene Silencing, RNA, Small Interfering, beta Catenin, Mice
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