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.
Highlights
We report here that siRNA prepared with traditional reduced-serum transfection media (Opti-MEM) are excluded at the matrigel boundary with limited spheroid/organoid internalization
SW1463 spheroids showing localization of control-Cy3 siRNA formed in Opti-MEM using RNAiMax at 2, 6 and 24 hours post transfection. (C) Representative confocal images of SW1463 spheroids showing localization of control-Cy3 siRNA formed in 10% serum using RNAiMax at 2, 6 and 24 hours post transfection
Summary graphs indicating the level of CTNNB1 mRNA in (D) 3D, and (E) 2D SW1463 cell cultures, at 48 hours post transfection with control or CTNNB1 siRNA complexes formed in Opti-MEM or 10% serum using RNAiMax
Summary
We report here that siRNA prepared with traditional reduced-serum transfection media (Opti-MEM) are excluded at the matrigel boundary with limited spheroid/organoid internalization. SiRNA formed and delivered using standard serum-containing culture medium gains rapid entry to both matrigel and spheroids/ organoids, and elicits a transient gene knockdown.
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