airway epithelial cell complete growth media  (PromoCell)

Journal: Scientific Reports

Article Title: High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

doi: 10.1038/s41598-021-94095-7

Figure Lengend Snippet: Summary detailing the primary normal human tracheobronchial epithelial cells (NHBEs) used to grow human airway tissue within the PREDICT96-ALI platform.

Article Snippet: To seed PREDICT96-ALI plates, NHBEs were thawed, counted, re-suspended in complete small airway epithelial cell growth media (SAGM; Lonza), 100 U/mL penicillin–streptomycin (Thermo Fisher), 5 μM ROCKi (Tocris), 1 μM A-83-01 (Tocris), 0.2 μM DMH-1 (Tocris), 0.5 μM CHIR99021 (Tocris) as described elsewhere (hereafter referred to as SAGM + 4i), and plated at 10,000 cells per device in a 3 μL volume directly onto the membrane.

Techniques:

Journal: Scientific Reports

Article Title: High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

doi: 10.1038/s41598-021-94095-7

Figure Lengend Snippet: Healthy PREDICT96-ALI airway model. ( A ) Schematic illustrating the configuration of the biomimetic airway model within the PREDICT96-ALI platform with associated microenvironmental features including differentiated cell populations (ciliated, goblet, basal and club cells) of the mature tissue, apical mucus and periciliary fluid present on mature human airway, and basolateral fluid flow to recirculate nutrients, remove waste products, and oxygenate the media. ( B ) Timeline detailing proliferation, differentiation, inoculation and processing (sample collection, fixation, imaging, etc.) of the PREDICT96-ALI airway tissue over 4–6 weeks. ( C ) Stains of ciliated (acetylated-tubulin, green; β-tubulin, red), basal (CK5, green), and goblet (Muc5ac, green) cells counterstained for nucleic acids (DAPI, blue) and actin (phalloidin, grey) at ×40 magnification shown, with a 50 µm scale bar. Donor B is featured as representative. ( D ) High-resolution confocal microscopy ×40 z-stack orthogonal image of the pseudostratified epithelium with approximately 4 cell layers and 50 µm thickness established after culture at 28 days at an ALI. Donor B is featured as representative. ( E ) Transepithelial electrical resistance (TEER) plotted over time for three human primary epithelial cell donor populations, showing that the same ultimate level of barrier function is reached, although the kinetics of barrier resistance are different.

Article Snippet: To seed PREDICT96-ALI plates, NHBEs were thawed, counted, re-suspended in complete small airway epithelial cell growth media (SAGM; Lonza), 100 U/mL penicillin–streptomycin (Thermo Fisher), 5 μM ROCKi (Tocris), 1 μM A-83-01 (Tocris), 0.2 μM DMH-1 (Tocris), 0.5 μM CHIR99021 (Tocris) as described elsewhere (hereafter referred to as SAGM + 4i), and plated at 10,000 cells per device in a 3 μL volume directly onto the membrane.

Techniques: Imaging, Confocal Microscopy

Journal: Scientific Reports

Article Title: High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

doi: 10.1038/s41598-021-94095-7

Figure Lengend Snippet: Healthy PREDICT96-ALI airway model with freshly harvested epithelial cells from research bronchoscopies. Panels illustrating establishment of mature tissue with pseudostratified morphology, approximately 30–50 µm, thick from freshly harvested airway epithelial cells obtained from living research bronchoscopy donor D. ( A–D ) ×40 images showing basal cell populations (CK5, green), goblet cells (Muc5AC, green), ciliated cells (acetylated tubulin, green; β-tubulin, red), and club cells (Scgb1a1, red; Scgb3a2, red) counterstained with nucleic acid (DAPI, blue) and actin (phalloidin, grey) with scale bar 100 µm, ( E ) ×10 phase contrast image of mature tissue at 28 days at an ALI with scale bar 100 µm, and ( F ) ×40 phase contrast image of mucus droplets (black arrows) on surface of mature tissue at 28 days at an ALI with scale bar 50 µm. Cells are characterized by robust ciliary beat (see supplementary video) and mucociliary flow.

Article Snippet: To seed PREDICT96-ALI plates, NHBEs were thawed, counted, re-suspended in complete small airway epithelial cell growth media (SAGM; Lonza), 100 U/mL penicillin–streptomycin (Thermo Fisher), 5 μM ROCKi (Tocris), 1 μM A-83-01 (Tocris), 0.2 μM DMH-1 (Tocris), 0.5 μM CHIR99021 (Tocris) as described elsewhere (hereafter referred to as SAGM + 4i), and plated at 10,000 cells per device in a 3 μL volume directly onto the membrane.

Techniques:

Journal: Scientific Reports

Article Title: High-throughput human primary cell-based airway model for evaluating influenza, coronavirus, or other respiratory viruses in vitro

doi: 10.1038/s41598-021-94095-7

Figure Lengend Snippet: Immunofluorescence staining and quantification of IAV-infected PREDICT96-ALI airway tissue. ( A ) Staining of the nucleoprotein (NP, green) for IAV, actin (phalloidin, grey), β-tubulin (red) and nucleic acids (DAPI, blue), including merged panels for all four stains, within PREDICT96-ALI airway tissue developed using freshly harvested epithelial cells from donor D at 48 h p.i., after 5 weeks ALI culture and following inoculation with A/California/04/09 H1N1 (MOI 0 [mock control], 0.1 or 1). Strong expression of IAV-NP is seen at 48 h p.i. at both MOI 0.1 and 1; noted here is that this is one field of view (FOV), and that there is more cytopathic effect at MOI = 1. Images captured are ×40, z-stacks, slice 25 of 48. Scale bar 50 µm. ( B ) Quantification of the mean fluorescence intensity (MFI) of the IAV-NP stain (green) or Alexa Fluor 488 signal, indicating significant increase in IAV replication at both 0.1 and 1 MOIs. N = 2 independent experiments with data presented from donor D as representative. Per experiment, N = 3 tissue replicates per donor, time-point and condition (MOI, anti-viral dose). One FOV was analyzed at N = 7 slices (at 5 slice intervals) for each condition. Statistical significance: **p ≤ 0.01 and ***p ≤ 0.001.

Article Snippet: To seed PREDICT96-ALI plates, NHBEs were thawed, counted, re-suspended in complete small airway epithelial cell growth media (SAGM; Lonza), 100 U/mL penicillin–streptomycin (Thermo Fisher), 5 μM ROCKi (Tocris), 1 μM A-83-01 (Tocris), 0.2 μM DMH-1 (Tocris), 0.5 μM CHIR99021 (Tocris) as described elsewhere (hereafter referred to as SAGM + 4i), and plated at 10,000 cells per device in a 3 μL volume directly onto the membrane.

Techniques: Immunofluorescence, Staining, Infection, Control, Expressing, Fluorescence