Three-dimensional (3D) human airway organoids were generated from adult stem cells isolated from the non-tumor lung tissue obtained from patients undergoing lung resection in the Department of Surgery at Queen Mary Hospital. Organoid cultures (approximately 100 µm in diameter) were extracted from droplets of Matrigel (Growth Factor Reduced Basement Membrane Matrix; Corning) by using Gentle Cell Dissociation Reagent (STEMCELL Technologies) and sheared by mechanical disruption with 1-mL pipettes to allow viruses to gain access to the apical and basolateral sides of the epithelium. Around 100–200 organoids were infected with each influenza virus at 106 pfu/mL for 1 h at 37 °C. Organoids were washed with culture medium three times, re-embedded in Matrigel, and plated in prewarmed 24-well suspension culture plates (Greiner). Once solidified, the Matrigel droplets were maintained in complete organoid medium38 (link), and incubated at 37 °C in 5% CO2. The viral titers in the culture supernatants were assessed at 1, 24, and 48 hpi by TCID50 assays in MDCK cells.
24 well suspension culture plates
Manufactured by Greiner
Sourced in Austria
The 24-well suspension culture plates are designed for culturing cells in suspension. They provide a uniform and consistent environment for the growth and maintenance of cells in liquid media.
Automatically generated - may contain errors
Lab products found in correlation
Growth factor reduced basement membrane matrix, by Corning (2 mentions)
Matrigel, by Corning (2 mentions)
Matrigel, by Greiner (1 mentions)
Gentle cell dissociation reagent (gcdr), by STEMCELL (1 mentions)
100 μm cell strainer, by BD (1 mentions)
Collagenase, by Merck Group (1 mentions)
Eclipse ts100 inverted routine microscope, by Nikon (1 mentions)
Cultrex growth factor reduced bme type 2, by Bio-Techne (1 mentions)
70 m cell strainer, by Corning (1 mentions)
Retronectin, by Takara Bio (1 mentions)
Phosphate buffered saline (pbs), by Fresenius (1 mentions)
24 well tissue culture plate, by Corning (1 mentions)
Kanamycin, by Merck Group (1 mentions)
Carbenicillin, by Merck Group (1 mentions)
5 protocols using 24 well suspension culture plates
1
3D Airway Organoid Influenza Infection
2
Generation of Lung Organoids from Human Tissue
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The generation of four lines ASC-derived human AOs was based on the previous protocol (14 (link)) with a success rate of 100%. Briefly, with ethical approval by the Institutional Review Board of the University of Hong Kong/Hospital Authority Hong Kong West Cluster (UW 13-364) and informed consent of the patients, small pieces of normal lung tissue (∼0.2–0.6 cm2 in size) adjacent to the diseased tissues were obtained from patients who underwent resection. The tissues were minced and digested with 2 mg/mL collagenase (Sigma Aldrich) for 1–2 h at 37 °C, followed by shearing using a glass Pasteur pipette (Drummond) and straining over a 100-μm cell strainer (FALCON). The resultant single cells were embedded in 60% Matrigel (Growth Factor Reduced Basement Membrane Matrix; Corning) and were seeded in 24-well suspension culture plates (Greiner Bio-One). After solidification, Matrigel droplets were maintained with AO culture medium (SI Appendix, Table S1 ) at 37 °C in a humidified incubator with 5% CO2. The organoids were passaged every 2–3 wk. The bright-field images of the organoids were acquired using a Nikon Eclipse TS100 Inverted Routine Microscope.
3
Breast Cancer Organoid Culture Protocol
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Organoids were cultured as previously described.13 (link) Fresh BC tissues cut into 1–3 mm3 pieces were digested in BC tissue digest medium (Table S1 Table S2 Table S3
4
Transduction of Cell Lines with Genetic Constructs
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The cell lines T2, K562, OCI-AML2 and OCI-AML3 were transduced with constructs encoding HLA-A3, HLA-A11, wild-type NPM1 (wtNPM1) or dNPM1 in combination with tNGFR or CD34 as markers. Cells were transduced in 24-well suspension culture plates (Greiner Bio-One, Kremsmünster, Austria) that had been coated with 30 µg/mL RetroNectin (TaKaRa Bio, Kusatsu, Japan) and blocked with 2% 200 g/L human serum albumin (Sanquin) in PBS (Fresenius Kabi, Bad Homburg, Germany). Retroviral supernatant was added and plates were centrifuged at 2000× g for 20 min at 4 °C. The retroviral supernatants were removed and 500 µL of cells, at a concentration of 400,000–600,000 cells/mL, was added to the wells. After overnight incubation, cells were transferred to 24-well tissue culture plates (Corning). The transduced cells were purified by MACS or FACS and their purity was assessed by FACS before their use in our experiments.
5
Evaluating Antimicrobial Efficacy of CyC Analogs
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This was performed in 24-well suspension culture plates (Greiner bio-one) as described in [13] . E. coli and P. aeruginosa were grown on LB agar medium at 37°C. All mycobacteria were grown at either 32°C (M. marinum) or 37°C (M. smegmatis, M. bovis BCG, M. abscessus S and R variants, and M. tuberculosis mc 2 6230) on Middlebrook 7H10 agar (BD Difco) supplemented with 10% OADC and 24 µg/mL D-panthothenate (M. tuberculosis mc 2 6230).
The wells were filled with 1 mL of the appropriate medium containing each of the CyC analogs at a single 30 µM final concentration. Each screening plate contained negative (DMSO) and positive (50 μM antibiotics) controls, as well as one well for sterility control (i.e., medium alone). For the 100% inhibition control we used 50 µM kanamycin (Sigma Aldrich) for M. marinum, M. abscessus, M. smegmatis, M. bovis BCG, M. tuberculosis and E. coli; and 50 µM carbenicillin (Sigma Aldrich) for P. aeruginosa. Each well was spotted with 10 µL of a bacterial culture at 5 × 10 5 cells/mL. Incubation time varied from 1 day to 2 weeks depending on the strain tested. The CyC compounds leading to a minimum of 50% growth inhibition were selected for subsequent minimal inhibitory concentrations (MIC) determination using the REMA assay.
The wells were filled with 1 mL of the appropriate medium containing each of the CyC analogs at a single 30 µM final concentration. Each screening plate contained negative (DMSO) and positive (50 μM antibiotics) controls, as well as one well for sterility control (i.e., medium alone). For the 100% inhibition control we used 50 µM kanamycin (Sigma Aldrich) for M. marinum, M. abscessus, M. smegmatis, M. bovis BCG, M. tuberculosis and E. coli; and 50 µM carbenicillin (Sigma Aldrich) for P. aeruginosa. Each well was spotted with 10 µL of a bacterial culture at 5 × 10 5 cells/mL. Incubation time varied from 1 day to 2 weeks depending on the strain tested. The CyC compounds leading to a minimum of 50% growth inhibition were selected for subsequent minimal inhibitory concentrations (MIC) determination using the REMA assay.
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