Abstract
Aims and Objectives: Transient transfection of whole organoids in 3D for the study of small RNA species such as miRNA and siRNA are hampered by a lack of specific and efficient gene manipulation tools. Here we aim to explore efficient small RNA delivery to whole organoids in 3D culture.
Methods: miRNA modulation was performed in patient iPSC-derived organoids dissociated to single cells within 3D matrigel using pre-miRs (PM) to overexpress miRNA and target gene expression was measured by qRT-PCR. Transfection of organoids either dissociated to single cells, as whole organoids within 3D Matrigel ‘in situ’ or as whole organoids released from Matrigel was explored using a fluorescently labelled siRNA in the presence and absence of foetal calf serum. Flow cytometry was used to determine transfection efficiency.
Results: Overexpression of PM145 in organoids re-plated as single cells in 3D Matrigel led to a reciprocal decrease in its known mRNA target, CFTR. siRNA transfection of whole organoids released from 3D Matrigel demonstrated the highest transfection efficiency of live cells at >90% followed by whole organoids ‘in situ’ (45.89%) and lastly organoids dissociated to single cells (24.45%). In all cases serum free media yielded a greater percentage of transfected cells as measured by FACS analysis.
Conclusions: Our preliminary data demonstrates modest but successful transfection of patient iPSC-derived organoid cultures ‘in situ’ in 3D matrigel using a commercial transfection reagent. Further development of these methods will facilitate miRNA:Target gene interaction studies to complement ongoing research in iPSC organoid platforms and inform delivery of future therapeutic strategies utilising small RNA.
Footnotes
Cite this article as Eur Respir J 2022; 60: Suppl. 66, 2368.
This article was presented at the 2022 ERS International Congress, in session “-”.
This is an ERS International Congress abstract. No full-text version is available. Further material to accompany this abstract may be available at www.ers-education.org (ERS member access only).
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