Development of a senescent organoid assay from an epithelial cell line could be a way to identify seno-active drugs in human emphysema - 20/03/24

Lung alveoli are essential for gas exchanges between air and blood. Alveoli can be destroyed as in emphysema, a disease in which an accumulation of senescent cells has been observed. No curative treatment is yet able to create new alveoli and regenerative treatments described in mice failed to be transferred successfully to patients with emphysema. This emphasizes the need to develop intermediate steps between mouse models and clinical trials for drug development in emphysema. Our aim was to develop 3D alveolar cell culture that could be used to screen senotherapeutic strategies, first organoids derived from alveolar epithelial and mesenchymal cell lines (H₄₄₁and MRC5) and then patient-derived alveolospheres from epithelial cells.

H₄₄₁ and MRC5 were cocultured in a drop of Matrigel in 24 trans-well and 96-well plates. AT1 (HT1-56) and AT2 (HT2-280) markers were assessed by immunofluorescence. The variability of organoid sizes and shapes were assessed over time to determine the optimal number of cells (MRC5 and H₄₄₁). We first used 24 trans-well plates, and next miniaturized with 96-well plates. These seeding conditions were compared between day 8 and day 16 of culture. Several senescence inducers were tested to trigger reproducible senescence in H₄₄₁: Doxorubicin (5nM to 50μM) and Etoposide (500nM to 100μM). Secondly, patient-derived alveolospheres were developed by isolating HT2-280 positive cells (AT2 cells) from human lung explants (Tata - Star protocol) and β-galactosidase was assessed to measure cell senescence.

Culturing H₄₄₁ in Matrigel led to the formation of spheroidal 3D structures expressing both AT1 and AT2 markers. Co-culturing H₄₄₁ with MRC5 in Matrigel increased by 150% the organoid number compared to H₄₄₁ alone but did not affect their size. In all culture conditions the organoid size increased between day 8 and day 16 of culture. Increasing the amount of H₄₄₁ initially seeded with MRC5 increased the organoid number (230% between 625 and 1250 H₄₄₁ and 770% between 625 and 5K H₄₄₁) but does not influence their size. The best seeding condition for the 24 trans-well plate was 1250 H₄₄₁ with 50K MRC5 and for the 96-well plate is 250 H₄₄₁ with 2500 MRC5. The maximum non-toxic dose was 25μM for etoposide and 5μM for doxorubicin. Both treatments induced senescence in H₄₄₁ cells. When the alveolospheres have been passed, the percentage of β-galactosidase positive organoids was 40% at first passage and 52% at second passage.

We developed two senescent alveolar organoids assay that could be used to test senotherapeutic drugs.