PneumaCult Apical-out AO Medium

STEMCELL Technologies offers this serum- and BPE-free cell culture medium for the differentiation of human primary bronchial epithelial cells (HBECs) or human airway epithelial cells (HAECs) into mature apical-out airway organoids. PneumaCult™ Apical-Out Airway Organoid Medium enables organoids that, when fully differentiated over 15 days, display outward-facing ciliated cells characteristic of the apical surface of the airway epithelium in vivo.

The complete kit combines a basal medium, a 10X supplement, and a 1000X supplement. The basal medium is stored at 2 - 8°C with a shelf life of 12 months from date of manufacture, the 10X supplement is stored at -20°C (stable for 2 years from manufacture), and the 1000X supplement is stored at -20°C (stable for 12 months from manufacture). A complete workflow involves first expanding HBECs or HAECs in PneumaCult™-Ex Plus Medium, then aggregating cells in an AggreWell™400 plate for 24 hours to 6 days, and finally differentiating aggregates in a tissue culture-treated 24-well flat-bottom plate for 9 - 14 days. By day 15, the resulting apical-out airway organoids display beating cilia suitable for downstream assays.

Applications include infectious disease modeling and high-throughput drug screening in three-dimensional (3D) in vitro model systems. Gene expression analysis shows upregulation of ciliated cell markers FOXJ1 and TUBB4B compared to ALI cultures, along with expression of ACE2 and TMPRSS2. Organoids comprise, on average, 62 cells (95% confidence interval of 6.77), demonstrating homogeneity at the cellular level. The organoids have been demonstrated to be susceptible to infection with Influenza A, Influenza B, Rhinovirus A-16, and Enterovirus-D68, supporting their use in respiratory virus research. The 10X supplement contains material derived from human plasma, with donors tested and found negative for HIV-1 and -2, hepatitis B, and hepatitis C. The 1000X supplement contains components dissolved in dimethyl sulfoxide (DMSO) and is light sensitive; minimize exposure to light during handling.