# Successful Delivery of Small Non-Coding RNA Molecules into Human iPSC-Derived Lung Spheroids in 3D Culture Environment

## TL;DR

Researchers developed a method to deliver small RNA molecules into human lung spheroids without disrupting their 3D structure, enabling gene expression studies in preclinical models.

## Contribution

A novel transfection method for small RNAs in 3D iPSC-derived lung spheroids that preserves their structure.

## Key findings

- Maximal siRNA delivery was achieved in whole spheroids released from Matrigel under serum-free conditions.
- Transfection of miR-29c and miR-21 mimics significantly reduced target protein levels in lung spheroids.
- SFTPC-siRNA transfection reduced SFTPC mRNA levels by 50% in iAT2 spheroids.

## Abstract

Background/Objectives: Spheroid cultures in Matrigel are routinely used to study cell behaviour in complex 3D settings, thereby generating preclinical models of disease. Ideally, researchers would like to modulate gene expression ‘in situ’ for testing novel gene therapies while conserving the spheroid architecture. Here, we aim to provide an efficient method to transfect small RNAs (such as microRNAs and small interfering RNAs, i.e., siRNAs) into human induced pluripotent stem cell (iPSC)-derived 3D lung spheroids, specifically alveolar type II epithelial cells (iAT2) and basal cell (iBC) spheroids. Methods: Transfection of iAT2 spheroids within 3D Matrigel ‘in situ’, whole spheroids released from Matrigel or spheroids dissociated to single cells was explored via flow cytometry using a fluorescently labelled siRNA. Validation of the transfection method was performed in iAT2 and iBC spheroids using siRNA and miRNA mimics and measurement of specific target expression post-transfection. Results: Maximal delivery of siRNA was achieved in serum-free conditions in whole spheroids released from the Matrigel, followed by whole spheroids ‘in situ’. ‘In situ’ transfection of SFTPC-siRNA led to a 50% reduction in the SFTPC mRNA levels in iAT2 spheroids. Transfection of miR-29c mimic and miR-21 pre-miR into iAT2 and iBC spheroids, respectively, led to significant miRNA overexpression, together with a significant decrease in protein levels of the miR-29 target FOXO3a. Conclusions: This study demonstrates successful transfection of iPSC-derived lung spheroids without disruption of their 3D structure using a simple and feasible approach. Further development of these methods will facilitate functional studies in iPSC-derived spheroids utilizing small RNAs.