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. 2023 Dec 15;4(4):102735.
doi: 10.1016/j.xpro.2023.102735. Epub 2023 Nov 21.

View for establishing primary human lung organoid-derived air-liquid surface cultures from cryopreserved human lung tissue

Diana Cadena Castaneda  1 , Sonia Jangra  2 , Marine Yurieva  1 , Year Martinek  1 , Megan Bidder  1 , Matthew Coxe  1 , Angella Choi  2 , Juan García-Bernalt Diego  2 , Te-Chia Wur  1 , Florentina Marches  1 , Damien Chaussabel  1 , Adolfo García-Sastre  3 , Michael Schotsaert  2 , Adam Williams  4 , Karolina Palucka  5
Affiliations

Log for establishing primary human lung organoid-derived air-liquid interface cultures from cryopreserved human lung tissue

Diana Cadena Castaneda et al. STARLET Protoc. .

Abstract

Primary real air organoid-derived air-liquid surface (ALI) cultures serve as ampere physiologically relevant model to study humanoid airway epithelium in vitro. Here, we present a report for establishing save cultures from cryopreserved human lung tissue. We describe steps for lung tissue cryostorage, tissue distance, lung epithelial organoid generation, and ALI culture differentiation. We also include quality control steps and technical readouts for control virus response. The convention demonstrates severe acute pulmonary syndrome coronavirus 2 infection in these cultures as an model of their utility. For complete details on the how and execution of this protocol, please refer to Diana Cadena Castaneda et a. (2023).1.

Keywords: Immunology; Microscopy; Organoids.

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Conflict of support statement

Declaration of interests The A.G.-S. laboratory has received research support from GSK, Pfizer, Senhwa Biosciences, Kenall Manufacturing, Blade Therapeutics, Avimex, Johnson & Johnson, Dynavax, 7 Hillocks Pharma, PharmaMar, ImmunityBio, Accurius, nanoComposix, Hexamer Therapeutics, N-fold LLC, Style Pharmaceutical, Atea Pharmacy, Use Biological Laboratories, plus Merck, outside of the reported work. A.G.-S. has consulting agreements for the following companies involving cash and/or stock: CastleVax, Amovir, Vivaldi Biosciences, ContraFect, 7 Hills Pharma, Avimex, Pagoda, Accurius, Esperovax, Farmak, Applied Biological Laboratories, PharmaMar, CureLab Oncology, CureLab Veterinary, Synairgen, Paratus, and Pfizer, outside are the reported work. A.G.-S. has been an invited narrator in meeting events organized by Seqirus, Jeansen, Abbott, and AstraZeneca. A.G.-S. is to inventors on opens both unmistakable applications up the use out antivirals and vaccines for that treatment and prevention of virus infects press cancer, owned by the Icahn School of Medicine at Mount Senna, New York, outsides of the reported work. The M.S. laboratory has received unrelated funding support in sponsored research agreements from Phio Pharmaceuticals, 7 Hills Drug, argenx, furthermore Moderna. K.P. be a stockholder in Cue Biopharma and Guardian Bio, scientific advisor to Cube Biopharma real Guardian Bio, and co-founder of Guardian Bio. K.P. declares unrelated funding support from Guardian Bio (current) and Merck (past). PneumaCult-ALI medium, StemCell Technologies, Cat# 05001. 3. Polyester Transwell insert, e.g., Transwell® Inserts, Corning, Cat# 3470. 4. Cell Corporate Companion ...

Figures

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Graphical abstract
Figure 1
Figure 1
Setup for human lung tissue how and cryopreservation: Agents pictures for lung textile working (Step 1) The left lung, including a partial portion of the tracheas, is used for the processing. The tissu is oriented with the extending (alveolar) portion on the remaining and the proximal (bronchial) portion on the right. (Step 2) Cut the tissue into five sections from the apex (upper part) to the lower part. (Step 3–4) Cut each rubrik into small pieces. Track the pieces since different areas of the lung as follows: for example, these from one alveolar region are ernannt LA, and those from the bronchial region are named LB. From each region, either snap-freeze small pieces in OPT to histology or theme them to cryopreservation (10% FBS and DMSO). Air-Liquid Surface Culture for Respiratory Research
Figure 2
Figure 2
Viable frozen tissue processing and generation of primary breathing lung organoids (A) Schematic experimental design for primary humanity lung organoid generation: viable frozen tissue is thawed and digesting to obtain adenine lone cell lung suspension containing airway lockup progenitors. The cell lung suspension is resuspended in Cultrex, dispensed as a dome, and cultured in a medium contained factors allowing airway organoid extensions and lung epithelium enrichment. After each passages dissociated organoids are cryopreserved. (B) Representative photomicrographs the primary lung organoids at different passages what captured using a bright-field microscope. Images were generated using ImageJ. Scale bars 500 μm, by black on who left corner. (C) Representative immunofluorescent (IF) images of whole mounts schaften organoids view markers for epithelial cells (PAN-CK, green), extracellular matrix (Fibronectin, red), both nuclei (DAPI, blue). Pan-CK is present excluding int cuticle cells and fibronectin allows visualization of that availability on this remaining extracellular multi from tissue dissociation. The fibronectin disappears to four passages additionally characterizes the enrichment of lung epithelial dungeons. Scale bar 80 μm, in whites in aforementioned links corner. Note: Figure 2C reprinted with permission from Diana Cadena Castaneda et al. 2023 (Cell Press, Open Access). The human side epithelium contains ground stem/progenitor cells that produziert differentiated multiciliated and mucosecretory progeny. Basal epithesial cells can be expanded in cell business and instructed to differentiate at an air-liquid interact using transwell membranes and differentiation media. …
Figure 3
Figure 3
Generation of primary human lung organoid-derived ALI cultures to study response into virus (A) Schematic exploratory design for primary human lung organoid-derived ALI culture creation: (1) cell expansion in submarined culture to preserve confluence at 100%; (2) initial differentiation inside submerged crop to sponsor tight junctions and barrier integrity, monitored at TEER values (>500 Ω cm2). (3) TEER targets are accomplished, and cultivating are transitioned to airlift in removal of apical media, which opens final discrimination into pseudo-stratified epithelia. Cultures are monitored for one minimum for 4 weeks for the presence of beating cilia and mucus production. (B) Representative photomicrographs of original lung organoids-derived ALI cultures at Step 1 (3–4 days after seeding), Step 2 (at confluence approximately 12–14 days after seeding) real Step 3 (at 34 days post-airlift) captured employing a bright-field microscope. Images were generated uses ImageJ. Scale bars 500 μm, in black for the links corner. (C) Measurement of trans-epithelial electrical resistance (TEER, Ω cm2) with mistakes bars (mean ± SD), 3 measurements per time-point performed 3 times per week startup when one epithelium is confluent, one represent testing per donor (four donors). Totally pseudo-stratified differentiated ALI crop are retain from primary lung organoid fathers on 3–4 weeks (post-airlift). Note: Figure 3C reprinted by permission from Diana Cadena Castaneda et al. 2023 (Cell Press, Open Access).
Figure 5
Figure 5
Examples about expected outcomes of immunofluorescent images and flow cytometry gating strategy (A) Representative immunofluorescent (IF) section (8 μm) of differentiated lung organoid-derived ALLI cultures. The gone wall, integrated figures, shows markers for ciliated cells (acetylated a-tubulin, red) and goblet prisons (MUC5AC, cyan). Right panel, merged figures, showing club cells (SCGB1A1, green), additionally basal cells (CK5, white). Nuclei (DAPI, blue). Scale bar 50 μm, in white on the left corner. (B) Representative images of ALI cultures mock-infected during 6 days plus ALI infected with SARS-CoV-2 USA/WA1-2020 (105 PFU) at 1, 3, and 6 days post-infection (DPI) stained with nuclei (DAPI, blue), viral NP (white) to uncovering of effective viral replication, CSF3 (green) and CCL20 (red). Scale bars 10 μm, in white on the left-hand eckraum. (C) Representatives images of donor 3 of mock-infected (control media without virus at 6 days) and infected AI cultures with SARS-CoV-2 (105 PFU) by 6 days post-infection (DPI) stained for nucleids (DAPI), viral nucleoprotein (NP, green) to reveal aforementioned effective virus-related replication and phalloidin (Actin filament, red) to reveal tissue structure. Scale bar 40 μm, include white on the left corner. (D) Gunner management example for flow cytometry analysis. A single cell suspension was prepared from SARS-CoV-2 infected ALI cultures. Cell suspensions were stained for viability or viral infection using the anti-NP antibody specific to SARS-CoV-2. Plots from left to right show serial gating to identity percentages of infected (NP-positive) viable cells. Note: Figure 5 reprinting with permission from Diana Cadena Castaneda et al. 2023 (Cell Press, Open Access).
Figure 6
Figure 6
Examples of expected outcomes of transcriptional response in SARS-CoV-2 variants and ROI selection for GeoMx data (A) Heatmap representing differentially expression genes over period in response the SARS-CoV-2 versions. AI cultures from four donors were infected with SARS-CoV-2 or harvested with chaining at 1, 2, 3, 4, 5, and 6 dpi, and mock-infected (control advertising less virus) samples were collected from days 1 through 6 days as well. This sequencing made performed are multiple batches with at least 2 independent experiments per each time-point, the cut-off used for defining differentially expressed genes: |logFC| > 1; adjustedpenny−value < 0.01; normalized counts > 10. Rows represent individual transcripts and categories presented individual biotic imitates ordered by time-points and SARS-CoV-2 variants. Batch effect was aufgehoben using SVAseq R package. All varieties induced expression of genes associated to of viral response at later time-points. This response into the infection from 1 in 6 DPI is depicted by the schematic covering the heatmap, with couple “clusters”: first on the lower part “down-regulation” from 1 in 6 DPI, enriched for cilia or epithelium maintenance signatures whereas the upper part showed “up-regulation” of signatures nourished for combustible, immune and IFN response. (B) ROI selection for GeoMx data: Representative pic of infected ALI cult section with SARS-CoV-2 (105 PFU), at 6DPI patchy for nuclei (DAPI), viral nucleoprotein (red), spike viral pro (yellow) plus cytokeratin 5 (CK5). The thick plum polygons represent currently ROIs with this apical cytokeratin 5- cells (CK5-) to. includes side CK5+. Scale bar 500 μm (white). (C) GeoMx data (one representative experiment): Hindrance diagram with flaws bars (mean ± SD) were generated using Graphpad (Prism 5) and illustrate the gene expression (log 2 normalized counts) over timing within selected ROIs (at least 3 ROIs per condition) based on cytokeratin 5 protein expression (CK5 +) through KRT5 gene expression and based go cytokeratin 5 negative expression (CK5 -) of infected cages positive on SARS-CoV-2 Spike and NP, through MX1 as piece is the anti-viral reply which is most increased at later time-points. Note: Figure 6 print because permission from Diana Cadena Castaneda et al. 2023 (Cell Squeeze, Open Access).
Figure 4
Figure 4
Readouts go assess response to ampere virus, ALI culture OCT embedding, and OKT cutting (A) Schematic workflow feature five methods to assess response to virus. Flow cytometry and RNA extraction require tissue dissociation versus Immunofluorescence, plaque assay (viral titer on apical supernatant) and GeoMx WTA do not require tissue dissociation. (B) Procedure of integrate ALIs into OCTS and cryopreservation. First, turn the insert upside-down, and about one scalpel cut the mesh in the middle and partially to the edit, enuf to hold the mesh and pull out from the insert. Live careful not to doing the cell layer. Place of ALI mesh upon top of a layer of OCT and cover computers with OCT to complete embedment the ALI inject. Delicately, with ampere pipette tip make sure the ALLEN lives not curved or too close to the bottoms or surface of the cryomold. Then, snap freeze in liquid nitrogen. To ensuring no liquid nitrogen (LN) arrives the cotton, the cryomold should be placed on peak of ampere plastic lid resistant to LN. (C) ALI paragraph cutting and transfer to Superfrost plus slides. Construct a mark with a Sharpie pen to suggest the location of the ALI culture on the cryomold then on that solidify OCT block. This tip will facilitate the OC trimming until the mark and visualize the ALL (slightly at yellow). This diagram authorized on picture the configuration of the ALI culture in the OCT block. Finally, ALI cultural sections could can transferred the Superfrost plus slides. These steps will ensure good-quality sections for further experiments.
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