Figure 1
Times and metrics of ALI cultures. (A) Days in cultures needed to reach 70–80% confluency in T75 flasks, 100% confluency on transwell inserts and full differentiation at ALI for adult (red; n = 5) and preterm (green, n = 5) pNECs. Data from two independent experiments of each proband are shown. (B) Cell and nucleus volumes were calculated from 3D reconstructed confocal images using Imaris imaging software. Based on the in-build “Cells” algorithm, the cellular volumes were calculated for each identified cell based on nuclear (DAPI) and actin/plasma membrane (Phalloidin) staining. Data are summarized from four independent experiments. (C) The number of basal cells, goblet cells and (D) epithelial thickness was measured from confocal images of adults (n = 3) and preterm infants (n = 3) using the Imaris imaging software. (E) Transepithelial electrical resistance (TEER) from fully differentiated cell culture inserts was assessed for adult (n = 5) and preterm (n = 5) pNECs. Data from two independent experiments of each proband are shown. (F) H&E stained sections of adult (left panel) and preterm (right panel) ALI cultures. (A–E) Boxes represent the 25th to 75th percentile and error bars indicate the 5th and the 95th percentile. Median values are represented by the box middle line. Black dots indicate outliers. Black triangles represent each individual data point. Normal distribution was determined with Shapiro-Wilk-test. Normal distributed data was analyzed using student’s t-test and not-normal distributed data was analyzed using Mann-Whitney test. **p < 0.01, ****p < 0.0001.
