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Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

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Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. / Morgan, R. G.; Chambers, A. C.; Legge, D. N.; Coles, S. J.; Greenhough, A.; Williams, A. C.

In: Scientific Reports, Vol. 8, No. 1, 7952, 21.05.2018.

Research output: Contribution to journalArticle

Harvard

Morgan, RG, Chambers, AC, Legge, DN, Coles, SJ, Greenhough, A & Williams, AC 2018, 'Optimized delivery of siRNA into 3D tumor spheroid cultures in situ', Scientific Reports, vol. 8, no. 1, 7952. https://doi.org/10.1038/s41598-018-26253-3

APA

Morgan, R. G., Chambers, A. C., Legge, D. N., Coles, S. J., Greenhough, A., & Williams, A. C. (2018). Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. Scientific Reports, 8(1), [7952]. https://doi.org/10.1038/s41598-018-26253-3

Vancouver

Morgan RG, Chambers AC, Legge DN, Coles SJ, Greenhough A, Williams AC. Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. Scientific Reports. 2018 May 21;8(1). 7952. https://doi.org/10.1038/s41598-018-26253-3

Author

Morgan, R. G. ; Chambers, A. C. ; Legge, D. N. ; Coles, S. J. ; Greenhough, A. ; Williams, A. C. / Optimized delivery of siRNA into 3D tumor spheroid cultures in situ. In: Scientific Reports. 2018 ; Vol. 8, No. 1.

Bibtex

@article{7016d28bafae462580553e2d0e4e5ace,
title = "Optimized delivery of siRNA into 3D tumor spheroid cultures in situ",
abstract = "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.",
keywords = "Cancer models, Gastrointestinal models",
author = "Morgan, {R. G.} and Chambers, {A. C.} and Legge, {D. N.} and Coles, {S. J.} and A. Greenhough and Williams, {A. C.}",
year = "2018",
month = "5",
day = "21",
doi = "10.1038/s41598-018-26253-3",
language = "English",
volume = "8",
journal = "Scientific Reports",
issn = "2045-2322",
publisher = "Springer Nature",
number = "1",

}

RIS - suitable for import to EndNote

TY - JOUR

T1 - Optimized delivery of siRNA into 3D tumor spheroid cultures in situ

AU - Morgan, R. G.

AU - Chambers, A. C.

AU - Legge, D. N.

AU - Coles, S. J.

AU - Greenhough, A.

AU - Williams, A. C.

PY - 2018/5/21

Y1 - 2018/5/21

N2 - 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.

AB - 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.

KW - Cancer models

KW - Gastrointestinal models

UR - http://www.scopus.com/inward/record.url?scp=85047320489&partnerID=8YFLogxK

U2 - 10.1038/s41598-018-26253-3

DO - 10.1038/s41598-018-26253-3

M3 - Article

VL - 8

JO - Scientific Reports

JF - Scientific Reports

SN - 2045-2322

IS - 1

M1 - 7952

ER -