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Optimizing miRNA transfection for screening in precision cut lung slices

Nowakowska, Joanna LU ; Gvazava, Nika LU ; Langwiński, Wojciech ; Ziarniak, Kamil ; da Silva, Iran Augusto N. LU orcid ; Stegmayr, John LU ; Wagner, Darcy E. LU orcid and Szczepankiewicz, Aleksandra (2024) In American Journal of Physiology - Lung Cellular and Molecular Physiology 327(5). p.712-723
Abstract

Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy... (More)

Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualized using live high-content fluorescence microscopy, followed by higher-resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2, we established that cellular membranes in PCLS are permeable in the initial 24 h of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 h after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared with other LNPs, with detection possible as low as 25 nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.

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author
; ; ; ; ; ; and
organization
publishing date
type
Contribution to journal
publication status
published
subject
keywords
miRNA, precision cut lung slices, transfection
in
American Journal of Physiology - Lung Cellular and Molecular Physiology
volume
327
issue
5
pages
712 - 723
publisher
American Physiological Society
external identifiers
  • scopus:85208091881
  • pmid:39254091
ISSN
1040-0605
DOI
10.1152/ajplung.00138.2024
language
English
LU publication?
yes
id
131b7aa2-0c44-402a-8f00-b10bec7597f0
date added to LUP
2024-12-10 13:44:25
date last changed
2025-06-11 03:49:18
@article{131b7aa2-0c44-402a-8f00-b10bec7597f0,
  abstract     = {{<p>Precision cut lung slices (PCLS) are complex three-dimensional (3-D) lung tissue models, which preserve the native microenvironment, including cell diversity and cell-matrix interactions. They are an innovative ex vivo platform that allows studying disease as well as the effects of therapeutic agents or regulatory molecules [e.g., microRNA (miRNA)]. The aim of our study was to develop a protocol to transfect PCLS with miRNA using lipid nanoparticles (LNPs) to enable higher throughput screening of miRNA, obviating the need for custom stabilization and internalization approaches. PCLS of 4 mm diameter were generated using agarose-filled rodent lungs and a vibratome. TYE665-labeled scrambled miRNA was used to evaluate transfection efficacy of six different commercially available LNPs. Transfection efficacy was visualized using live high-content fluorescence microscopy, followed by higher-resolution confocal fluorescence microscopy in fixed PCLS. Metabolic activity and cellular damage were assessed using water-soluble tetrazolium salt (WST-1) and lactate dehydrogenase (LDH) release. Using a live staining kit containing a cell membrane impermeant nuclear dye, RedDot2, we established that cellular membranes in PCLS are permeable in the initial 24 h of slicing but diminished thereafter. Therefore, all transfection experiments occurred at least 24 h after slicing. All six commercially available LNPs enabled transfection without inducing significant cytotoxicity or impaired metabolic function. However, RNAiMAX and INTERFERin led to increases in transfection efficacy as compared with other LNPs, with detection possible as low as 25 nM. Therefore, LNP-based transfection of miRNA is possible and can be visualized in live or fixed PCLS, enabling future higher throughput studies using diverse miRNAs.</p>}},
  author       = {{Nowakowska, Joanna and Gvazava, Nika and Langwiński, Wojciech and Ziarniak, Kamil and da Silva, Iran Augusto N. and Stegmayr, John and Wagner, Darcy E. and Szczepankiewicz, Aleksandra}},
  issn         = {{1040-0605}},
  keywords     = {{miRNA; precision cut lung slices; transfection}},
  language     = {{eng}},
  number       = {{5}},
  pages        = {{712--723}},
  publisher    = {{American Physiological Society}},
  series       = {{American Journal of Physiology - Lung Cellular and Molecular Physiology}},
  title        = {{Optimizing miRNA transfection for screening in precision cut lung slices}},
  url          = {{http://dx.doi.org/10.1152/ajplung.00138.2024}},
  doi          = {{10.1152/ajplung.00138.2024}},
  volume       = {{327}},
  year         = {{2024}},
}