Portal de Periódicos da CAPES

High-Throughput Culture Method of Induced Pluripotent Stem Cell Derived Alveolar Epithelial Cells Using Floating Matrigel Droplets

2021; Elsevier BV; Volume: 40; Issue: 4 Linguagem: Inglês

10.1016/j.healun.2021.01.1866

1557-3117

David Becerra, Sydney Jeffs, Tong Wu, Harald C. Ott,

Electrospun Nanofibers in Biomedical Applications

Purpose Induced pluripotent stem cell derived alveolar epithelial cells (iPSC-AECs) are a patient specific cell source for bio-engineering of human pulmonary epithelium. Disease modeling and therapeutic applications require cost effective and technically feasible differentiation and expansion protocols. Current culture protocols are labor and resource intensive and not scalable for large animal or human studies. We developed a scalable culture protocol switching from adherent hydrogel drops to suspension culture. Methods Genetically modified iPSCs with fluorescent reporters for Nkx2.1 and SPC underwent differentiation in a stepwise culture protocol that mimics lung development. Cells were purified, sorted and embedded in a liquid Matrigel precursor. Homogenous liquid precursor was added to culture plates to form adherent droplets or to a silicone mold to form cell laden Matrigel spheroids which were subsequently transferred to spinner flasks with media as floating droplets. After culture, monolayer spheres of iPSC-AECs were digested and trypsinized to form single cell suspensions. Equal numbers of iPSC-AECs from the two culture conditions were seeded into decellularized lung scaffolds. Results iPSC-AECs cultured in floating droplets were more proliferative than adherent droplets, with significantly higher total cell counts and Ki67 expression. Equivalent expression of the distal lung markers Nkx2.1, SPC, and AQP5 was observed for both culture conditions in isolated iPSC-AECs and iPSC-AECs from recellularized lung scaffolds. Similar histologic appearance was appreciated on lungs recellularized from both culture groups. The floating droplet method was more time and cost efficient. Conclusion We report a simple, scalable, new culture method for iPSC-AECs with stable cellular phenotype that could support large animal and human studies. Our protocol enhanced proliferation and reduced labor requirements compared to previously reported methods.