Background and aim: Precision cut lung slices (PCLS) are complex 3D lung tissue models, which preserves the native microenvironment of the lung, including cell-matrix interactions. It is an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules, for example miRNA. The aim of our study was to develop a protocol to transfect PCLS with miRNA, which can be a step forward in the research of miRNA-based therapies.  

Methods: We administered agarose (1,5 and 2%) intratracheally to inflate mouse lungs and generated 4mm diameter PCLS using a vibratome and biopsy punch. TYE665 labelled miRNA was used to evaluate the transfection efficacy of 6 transfection reagents (RNAiMAX, Lipofectamine 3000, INTERFERin, TransIT-X2, TransIT-siQuest, ViaFect) and 2 miRNA concentrations (100 nM and 50 nM), with empty transfection reagents and nontransfected PCLS as controls. Transfection efficacy was visualised using confocal fluorescence microscopy and metabolic activity and cellular damage was assessed with the water soluble tetrazolium salt WST1 and lactate dehydrogenase (LDH) release. 

Results: We were able to visualise transfected miRNA in all samples and with all transfection reagents. No significant differences were detected in LDH release with either transfection reagent or miRNA itself.  We observed enhanced transfection efficacy with 50nM of miRNA using RNAiMAX and INTERFERin.  

Conclusion: It is possible to transfect PCLS with miRNA. TYE665 is an appropriate dye to label miRNA in this model. Future studies will focus on optimizing the miRNA concentration in the model and transfecting with miRNA of therapeutic interest.