Journal: Biofabrication

Article Title: Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform

doi: 10.1088/1758-5090/ac32a5

Figure Lengend Snippet: Immunofluorescence (IF) of small airway epithelial population at the epithelial level (a) and at the epithelial-endothelial junction (c), (d). (a) DAPI (blue), α-tubulin (green), MUC5AC (yellow), CK5 (magenta). (b) xyz-view of DAPI (blue), α-tubulin (green), MUC5AC (yellow), and CK5 (magenta) co-stain to show pseudostratified columnar epithelium structure of the small airway layer. (c) maximum projection of epithelial layer only. α-tubulin (green), ACE2 (magenta) along the surface/higher z-stacks of small airway epithelium. (d) maximum projection at vascular layer. DAPI (blue), CD31 (yellow), ACE2 (magenta) along vasculature layer/lower z-stacks of small airway-capillary constructs. (e) live perfusion of small airway-capillary construct at timepoint t=0 min. The dye was introduced into the reservoirs of the left parent channel (input channel/vessel) and the white arrows indicate the entry point from the vascular network into the output channel/vessel. Taken with Opera Phenix. Scale bar = 100 μm unless otherwise indicated in images.

Article Snippet: For the 3D SAEC-capillary construct, AF700-conjugated MUC5AC (Novus Biologicals, NBP2–32732AF700), PE-conjugated cytokeratin 5/8 (Novus Biologicals, NBP2–47824PE), and AF488-conjugated α-tubulin (Cell Signaling Technology, 8058S) were used to identify small airway epithelial cells.

Techniques: Immunofluorescence, Staining, Construct

Journal: Biofabrication

Article Title: Development of human-derived, three-dimensional respiratory epithelial tissue constructs with perfusable microvasculature on a high-throughput microfluidics screening platform

doi: 10.1088/1758-5090/ac32a5

Figure Lengend Snippet: Flow cytometric evaluation of cell types in 3D respiratory tissue construct and table with cluster frequencies. (a) bivariate plot of AQP5 and Sp-C in 3D alveoli-capillary construct. (b) bivariate plot of AQP5 and Sp-C on transwell platform, alveolar monoculture. (c) bivariate plot of AQP5 and Sp-C in submerged T-75 alveolar monoculture. (d) bivariate plot of a-tubulin and MUC5AC in 3D small airway-capillary construct. (e) bivariate plot of α-tubulin and MUC5AC on transwell platform, small airway monoculture. (f) bivariate plot of α-tubulin and MUC5AC in submerged, T-75 small airway culture. Grey = negative control; pink – stained sample. (g) bivariate plot of CD31 and vimentin in 3D respiratory construct. (h) bivariate plot of CD31 and vimentin of submerged T-75 monoculture for endothelial cells, fibroblasts and pericyte, contour plots of cell types overlaid. (i) Stacked column graph of cell populations from (a)-(f). VAMC = vascularized alveolar multi-chip; VBMC = vascularized bronchiolar multi-chip; AEC = alveolar epithelial cells; SAEC = small airway epithelial cells; TW = transwell. Blue = endothelial cells; red = fibroblasts; black = pericytes. FlowJo for compensation matrix calculation and gating analysis.

Article Snippet: For the 3D SAEC-capillary construct, AF700-conjugated MUC5AC (Novus Biologicals, NBP2–32732AF700), PE-conjugated cytokeratin 5/8 (Novus Biologicals, NBP2–47824PE), and AF488-conjugated α-tubulin (Cell Signaling Technology, 8058S) were used to identify small airway epithelial cells.

Techniques: Construct, Negative Control, Staining