Mar 08, 2026

Public workspacePrecision Cut Lung Slices (PCLS) as ex vivo Human Lung Tissue Model of Cellular Senescence Assessed by Multi-Omic Modalities

DOI
https://dx.doi.org/10.17504/protocols.io.3byl46m42go5/v1
  • sadiya shaikh1,
  • Heidie Huyck2,
  • Ariana Pitonzo3,
  • Irfan Rahman2,
  • Gloria Pryhuber1
  • 1University of Rochester Medical Center;
  • 2University of Rochester;
  • 3URMC
  • URMC Pryhuber Lab
  • Cellular Senescence Network (SenNet) Method Development Community
Gloria S Pryhuber
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DOI: https://dx.doi.org/10.17504/protocols.io.3byl46m42go5/v1
Protocol Citation: sadiya shaikh, Heidie Huyck, Ariana Pitonzo, Irfan Rahman, Gloria Pryhuber 2026. Precision Cut Lung Slices (PCLS) as ex vivo Human Lung Tissue Model of Cellular Senescence Assessed by Multi-Omic Modalities. protocols.io https://dx.doi.org/10.17504/protocols.io.3byl46m42go5/v1
License: This is an open access protocol distributed under the terms of the Creative Commons Attribution License,  which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited
Protocol status: Working
We use this protocol and it's working
Created: December 13, 2025
Last Modified: March 10, 2026
Protocol Integer ID: 234889
Keywords: Precision Cut Lung Slices, PCLS, Senescence, Irradiation, Senolytic, Fisetin, Luminex multiplex cytokine immunoassay, NanoString nCounter transcriptomic profiling, RNA extraction, efficacy of senescence inducer, senescence inducer, cellular senescence, induced senescence, induction of senescence, functional responses of lung tissue, secretome profiling via luminex, ex vivo human lung tissue model, following senescence induction, senescence induction, senescence, application of senolytic drug treatment, preservation of pcls tissue, senolytic drug treatment, secretome profiling, senolytic compound, pharmacological agent, secretory phenotype, precision cut lung slice, associated secretory phenotype, pcls tissue, multiplex cytokine immunoassay, cut lung slice, targeting gene, dna damage response, pcls supernatant, lung slice
Funders Acknowledgements:
NIA/NIH TriState SenNET Lung and Heart Tissue Mapping Consortium
Grant ID: NIH U54 AG075931
NHLBI LungMAP BioRepository for Investigation of Diseases of the Lung (BRINDL)
Grant ID: NIH U01 HL148861
Abstract
This protocol outlines the experimental workflow for evaluating the efficacy of senescence inducers and senotherapeutic / senolytic compounds in modulating the senescence-associated secretory phenotype (SASP) using precision-cut lung slices (PCLS) as an ex vivo model system. PCLS retain the native tissue architecture and cell-type complexity of the lung, making them a relevant multi-cellular platform to study spatial and functional responses of lung tissue to pharmacological agents. Version 1 of the protocol outlines:
  • Ex vivo induction of senescence by irradiation of PCLS using defined doses (e.g., 5–10 Gy) to model DNA damage-induced senescence and inflammatory activation.
  • Application of senolytic drug treatment (e.g., Dasatinib, Quercetin and Fisetin, or other candidate compounds) administered after recovery from irradiation.
  • Collection of PCLS supernatants for secretome profiling via Luminex multiplex cytokine immunoassays, focused on senescence-associated secretory phenotype (SASP) factors.
  • Recovery and preservation of PCLS tissue following senescence induction / inhibition for 1) RNA extraction and NanoString nCounter transcriptomic profiling, targeting genes involved in senescence, DNA damage response, and immune modulation, 2) collection of supernatants for secretome profiling via Luminex multiplex cytokine immunoassays, and 3) fixation, embedding and sectioning for histochemistry, mutiplexed immunofluorescence (MxIF) and/or spatial transcriptomics.
Troubleshooting
Safety warnings
Standard Universal Precautions should be utilized when working with the human PCLS tissue and fluids as they are unfixed and may carry pathogens. Personal protective equipment (PPE) should be utilized to reduce the likelihood of exposure to any known or unknown pathogens within the sample. Appropriate biosafety measures, generally BSL-2 at minimum, are followed in any manipulation of these human tissues or fluids originating from them.

Always follow institutional radiation safety protocols when operating or accessing the irradiator. Certified personnel are needed to run the radiation source.
Ethics statement
The protocols.io team notes that research involving animals and humans must be conducted according to internationally-accepted standards and should always have prior approval from an Institutional Ethics Committee or Board.
Before start
Preparation and storage of the human PCLS used in this protocol are detailed in 623.2.HTC_Precision_Cut_Lung_Slices V.2 (dx.doi.org/10.17504/protocols.io.4r3l2o26xv1y/v2.), The PCLS were stored individually in 1 ml freezing media made up of 10% (v/v) of DMSO in filtered FBS.
Day 1 - Preparation of frozen PCLS for in vitro experiment
Carefully retrieve, thaw and culture the PCLS
Remove PCLS vial from liqN2 freezer. Keep on Dry Ice until thawing can occur. Thaw by holding the freezer vial between fingers or briefly in a 37deg C water bath until just thawed- keep this thaw time to minimum.
Transfer individual PCLS into wells of a 6-well Tissue Culture plate containing 5 ml pre-warmed culture medium (DMEM+10%FBS+1% Antibiotic/Antimycotic).
Wash x 3 in 5 ml Culture Media then place in 24-well Tissue Culture plate with 1ml fresh media before placing in 37 deg C incubator with 5% CO2.
Allow slices to stabilize for approximately 24 hours in a 37°C, 5% CO₂ incubator.
This recommendation is based on observed time needed for recovery of WST-1 mitochondrial dehydrogenase activity.
Day 2 - Use of PCLS to Study Induction and Modification of Senescence
Administer experimental senescence inducer/senolytic/senomorphic treatment (Senotherapeutics (Inhibitors/Eliminators) to the PCLS
Experimental protocol may vary the timing, dose and duration of senescence inducer/senolytic/senomorphic treatment.
Example: Senescence Inducers (Pro-senescence Agents): Chemotherapy Agents (e.g., Doxorubicin, Cisplatin, Etoposide), Genotoxic Stress/Radiation (e.g., Ionizing radiation, DNA-damaging agents, Oncogenes: Oncogenic BRAF ( e.g., BRAF V600E).
Example: Senolytics (Kill senescent cells): Dasatinib, Quercetin, Navitoclax (ABT-263).
Example Senomorphics/Senostatics (Inhibit SASP/Function): Metformin, Rapamycin, JAK inhibitors, Antioxidants.
Add senolytic drugs directly to the culture medium to achieve final concentrations.
Include DMSO or other carrier-treated wells as vehicle controls.
Use single agents or combination treatment depending on experimental design.
Incubate treated PCLS at 37°C, 5% CO₂ in medium +/- at air-liquid interface
For long treatments for senescence-related study cultures, we cultured for 7 days. May choose to refresh media to maintain nutrient levels.
Day 5-7 - Collection of Samples for Analysis
Supernatant Collection for Luminex or Similar Assay
Carefully transfer supernatants (~400 µL) from each well to labeled low-binding tubes.
Centrifuge briefly (e.g., 5 min at 500 x g) to remove any debris.
Aliquot and freeze at –80°C until Luminex or similar analysis.

Isolation for single cell RNA sequencing
Our detailed protocol for isolation of single cells from these agarose inflated PCLS utilized in RNA sequencing can be found at "Production of High-Purity Cell Suspensions from Human Precision Cut Lung Slices for Single-Cell RNA Sequencing" https://dx.doi.org/10.17504/protocols.io.3byl4q3q8vo5/v1
Tissue Collection for RNA Isolation
Transfer each PCLS into a 1.5 mL tube with 350 µL RLT buffer or Trizol.
Homogenize using pellet pestle or motorized homogenizer.
Proceed with RNA isolation using the RNA kit of choice.
Elute in 15–30 µL nuclease-free water.
Measure RNA concentration and integrity (Nanodrop/Qubit and Bioanalyzer or TapeStation).
PCLS Preservation and Preparation for Immunohistochemistry.
Carefully transfer a PCLS section from the culture dish well onto a biopsy foam pad placed inside of a cassette, ensuring the piece lays flat. Place another foam pad on top and close the cassette lid, sandwiching the section so that it doesn't move during processing.
Place the cassettes into 10% NBF and at 4°C overnight, up to 24 hours.
After fixation, desalt and dehydrate the tissue by first rinsing the cassettes for 20 minutes in 1x PBS with agitation on a rotating platform at room temperature, followed by successive 20 minute agitations in 30% EtOH, 50% EtOH, then 70% EtOH. The cassettes should then be stored in the 70% EtOH at 4°C overnight until further processed.
From 70% EtOH, place the cassettes into the metal basket, and put the basket into the VIP processor’s retort.
Process using the "SHORT" program on the VIP Processor
70% Ethanol – 15 min at 40°C
80% Ethanol – 15 min at 40°C
95% Ethanol – 15 min at 40°C
95% Ethanol – 15 min at 40°C
100% Ethanol – 15 min at 40°C
100% Ethanol – 20 min at 40°C
Xylene – 20 min at 40°C
Xylene – 20 min at 40°C
Paraffin – 15 min at 60°C
Paraffin – 30 min at 60°C
Paraffin – 15 min at 60°C

After processing, PCLS sections should be embedded into paraffin blocks by taking them out of the cassettes and placing them into the metal embedding mold as they were in the cassette, ensuring the piece of tissue is still laying flat.
Cool molds on cold plate until paraffin is fully solid, then remove blocks and store at room temperature until ready to cut.
Section the paraffin embedded PCLS tissue onto slides (TruBond 380 Adhesive Microscope Slides for CODEX) in the following way:
  1. Fill a hot water bath with DEPC treated water and set to 45°C
  2. Using a microtome, trim the block carefully at 10µm until tissue is fully exposed. Place block on ice for 10 minutes to get cold.
  3. Section the block at 5 µm thickness, creating a ribbon of sections. 4 µm also an option.
  4. Place the ribbon onto the hot water bath, then pick up the desired sections onto glass slides in serial order, keeping track of order and placement. More than one tissue portion may be placed on each slide, staying within imaging area of imaging assayed planned.
  5. Dry the slides upright overnight until fully dry, then store in a slide box at room temperature until staining with H&E or CODEX.
Day 7+: Assays
Luminex Assay for SASP Factors
Retrieve stored supernatent samples
o Thaw supernatants on ice and dilute if necessary.
o Set up the Luminex plate according to kit instructions.
o Run samples and standards in technical duplicates.
o Analyze using Luminex reader FLEXMAP 3D.
o Quantify cytokines using provided standard curves.
NanoString nCounter Assay
Set Up Hybridization
o Use 50–100 ng of purified RNA per sample.
o Hybridize RNA with NanoString Reporter and Capture probes for 16hours at 65°C.
o Commercial or customized panel can be used
Post-Hybridization Processing
o Load reactions into the NanoString Prep Station for purification and immobilization.
o Scan samples using the nCounter Digital Analyzer.
Data Analysis: Normalize and analyze data using nSolver software.
Assess expression of senescence and immune-related transcripts.
Compare treated vs. control samples for differential SASP gene expression.
Multiplexed Immunofluorescence (MxIF) and Histology Imaging
Slides containing FFPE PCLS sections can be imaged using a Phenocycler Imaging System, CODEX MxIF Protocol followed by H&E stain as described in 813.1 Multiplexed Immunofluorescence Phenocycler-Fusion‱ Imaging of FFPE Lung Sections V.3 https://dx.doi.org/10.17504/protocols.io.6qpvr38dpvmk/v3
Experimental Protocols for TriState SenNet URMC data submissions.
Experiment #1. Pediatric Lung PCLS Model of Irradiation-Induced Senescence and Senolytic Intervention
See Data in Collection on SenNet Data Portal (doi: pending)

Day 2 - Ex vivo Irradiation of PCLS to induce Senescence
Transfer PCLS to site for delivery radiation dose as determined by experimental protocol.
o Transfer sealed plates to the irradiation facility using a sterile container or biosafety-approved transport method.
o Maintain plates at room temperature during transport. Minimize delays.
Recommended to transfer control plates to irradiation facility alongside those plates to be irradiated to control for changes in temperature, etc out of the incubator for the time needed to irradiate.
o Place the plate in the Cesium-137 irradiator chamber.
Caution: Certified personnel are needed to run the radiation source.
o Deliver a defined dose of ionizing radiation (commonly 5 Gy or 10 Gy) depending on experimental design. 10 Gy was used in this experiment.
o Assure that the Exposure is uniform across the entire plate
Post-irradiation recovery
o Immediately return the PCLS to a 37°C, 5% CO₂ incubator.
o Allow the tissue to recover for 4–6 hours before proceeding to senolytic treatment.
Timing of this step may be modified depending on experimental question.
o Optionally, extend recovery to 24 hours to assess delayed transcriptional or secretory responses.
Addition of Senolytic Treatment
Added senolytic drug to final concentration, directly to the culture medium, 48 hours after irradiation.
  1. Fisetin (Fisher Scientific, AC119150010) was reconstituted in DMSO and added to one control PCLS well and one irradiated PCLS well, to final concentration of (20 µM)
Incubated treated PCLS at 37°C, 5% CO₂ for 7 days with media changes every 2 days..
Day 5-7: Collection of Samples for Luminex, RNA Extraction and Immunohistochemistry
Samples were collected at day 7 post-treatment for downstream analyses.
Luminex assay for SASP factors
o Supernatant was collected, aliquoted and frozen at –80°C until Luminex assay as described in Step 7.
RNA Transcript Analysis
o PCLS were collected for RNA Isolation and NanoString nCounter Assay as described in Step 8.

Multiplexed Immunofluorescence (MxIF) Imaging
o PCLS were collected for MxIF analysis using our CODEX protocol and Phenocycler Fusion Imaging system
o The details of antibodies and protocol used on these sections is reported in the SenNet Portal.
Protocol references
  1. Martin, C., Uhlig, S., and Ullrich, V. (2001). Cytokine-Induced Bronchoconstriction in Precision-Cut Lung Slices Is Dependent upon Cyclooxygenase-2 and Thromboxane Receptor Activation. Am. J. Respir. Cell Mol. Biol. Vol 24, 139-145.
  2. Alsafadi, H. Et al (2017). An ex vivo model to induce early fibrosis-like changes in human precision-cut lung slices. Am. J. Physiol. Lung Cell Mol. Physiol. Vol 312 L896-L902.
  3. Neuhaus V. Et al (2018). Assessment of the Cytotoxic and Immunomodulatory Effects of Substances in Human Precision-cut Lung Slices. J. Vis. Exp. Vol 135, e57042.
  4. Alsafadi HN, Uhl FE, Pineda RH, Bailey KE, Rojas M, Wagner DE, Konigshoff M. Applications and Approaches for Three-Dimensional Precision-Cut Lung Slices. Disease Modeling and Drug Discovery. Am J Respir Cell Mol Biol. 2020;62(6):681-91. Epub 2020/01/29. doi: 10.1165/rcmb.2019-0276TR. PubMed PMID: 31991090; PMCID: PMC7401444.
  5. Lehmann M, Krishnan R, Sucre J, Kulkarni HS, Pineda RH, Anderson C, Banovich NE, Behrsing HP, Dean CH, Haak A, Gosens R, Kaminski N, Zagorska A, Koziol-White C, Metcalf JP, Kim YH, Loebel C, Neptune E, Noel A, Raghu G, Sewald K, Sharma A, Suki B, Sperling A, Tatler A, Turner S, Rosas IO, Van Ry P, Wille T, Randell SH, Pryhuber G, Rojas M, Bourke J, and Königshoff M. Precision-Cut Lung Slices: Emerging Tools for Preclinical and Translational Lung Research: An Official American Thoracic Society Workshop Report. Am J Respir Cell Mol Biol. 72: 16-31, 2024. doi.org/10.1165/rcmb.2024-0479ST PubMed: 39499861