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A7635 - Utility of Perfluorochemical Liquids for miRNA Transfection into Human Alveolar Type II Cells
Author Block: M. Marcinkiewicz1, S. T. Baker1, J. Wu1, Y. Tian2, S. Bolla3, C. Mandapati3, F. Salton4, M. Confalonieri4, N. Marchetti3, G. J. Criner3, M. R. Wolfson1; 1Physiology, Thoracic Medicine and Surgery, Center for Inflammation and Translational Lung Research, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, United States, 2Pharmacology, Center for Translational Medicine, Lewis Katz School of Medicine at Temple University, Philadelphia, PA, United States, 3Thoracic Medicine and Surgery, Center for Inflammation and Translational Clinical Lung Research, Lewis Katz School of Medicine at Temple University, Temple University Hospital, Philadelphia, PA, United States, 4University Hospital of Trieste, Trieste, Italy.
Rationale: Micro (miRNA) therapy for lung tissue repair is gaining increasing attention; however, current viral and nonviral delivery systems to distal lung epithelium are challenged by mucociliary clearance, barriers of the dense airway glycocalyx, and collectins function of lung surfactant and respective proteins. Perfluorochemical (PFCs) liquids, chemically inert media with high respiratory gas solubility and low surface tension, can be homogenously distributed in the lung, provide lung mechanoprotective and cytoprotective, and have been demonstrated as effective gene delivery vehicles. We have developed a method of cryopreserving ovine lung, generated alveolar epithelial type II (ATII) cells from this tissue with comparable phenotype to cells isolated from fresh tissue (PLos One 11(3)e0152027, 2016) and are applying this method of cryopreservation to human lung to extend our biobank. Here, we utilized human (h) ATII cells, isolated from fresh (Fr) or cryopreserved (Cryo) tissue, to test the hypotheses that PFCs can be useful transfection agents for miRNAs into hATII cells with efficiency related to physicochemical properties of PFC liquids.
Methods: Primary hATII cells were isolated from Fr or Cryo lung tissue obtained from the same deidentified organ donors whose lungs, not suitable for transplantation were donated for medical research. Cells were grown for 6 days, initially as submerged followed by 4 days air-liquid interface cell culture, then incubated with either 100% perfluoro-octylbromide (PFOB) or 25%/75% PP2/PP9 (MIX) each containing miRNA302b mimic at 10nM for 24 hrs, lysed and analyzed for the cell cycle inhibitor gene expression P21/CDKN1A (target gene of miRNA302b) and SP-B protein by qPCR. Non-transfected and PFC alone exposed cells served as respective controls.
Results: P21/CDKN1A gene expression was significantly lower than respective controls in hATII cells, from both Fr and Cryo tissue, transfected with miRNA302b mimic suspended in either PFC. Independent of tissue source, there was a PFC-dependent effect with greater inhibition by PFOB than mix. SP-B protein expression and cell viability were similar.
Conclusions: These data reflect that PFC liquids alone are effective miRNA to hATII cell transfection agents. Similar results in cells from cryopreserved vs fresh tissue support the use of cryopreservation methodology to expand our biobank. Given the efficacy of PFCs to support gas exchange and delivery of biologics to the lung, this study presents an alternative transfection method for safe and relatively rapid delivery system of miRNAs to the lung, in vivo without potential complications of existing transfection vehicles. NIH R01-HL111348, R00-HL132115 (Y.T.), DOD/ONR N000141210810 (MRW).