US 12,391,926 B2
Multi-layer airway organoids and methods of making and using the same
Sean V. Murphy, Winston-Salem, NC (US); and Anthony Atala, Winston-Salem, NC (US)
Assigned to Wake Forest University Health Sciences, Winston-Salem, NC (US)
Filed by Wake Forest University Health Sciences, Winston-Salem, NC (US)
Filed on Apr. 7, 2021, as Appl. No. 17/224,191.
Application 17/224,191 is a division of application No. 15/768,100, granted, now 11,001,811, previously published as PCT/US2016/056947, filed on Oct. 14, 2016.
Claims priority of provisional application 62/404,931, filed on Oct. 6, 2016.
Claims priority of provisional application 62/242,611, filed on Oct. 16, 2015.
Prior Publication US 2021/0222131 A1, Jul. 22, 2021
Int. Cl. C12N 5/071 (2010.01); C12M 1/00 (2006.01); C12M 3/00 (2006.01); C12M 3/06 (2006.01)
CPC C12N 5/0697 (2013.01) [C12M 21/08 (2013.01); C12M 23/16 (2013.01); C12M 23/22 (2013.01); C12N 5/0688 (2013.01); C12N 2502/27 (2013.01); C12N 2502/28 (2013.01); C12N 2533/54 (2013.01); C12N 2533/90 (2013.01)] 20 Claims
 
1. A method of making a lung organoid, the method comprising:
depositing an endothelial cell layer comprising mammalian endothelial cells onto a first side of a porous membrane;
depositing a stromal cell layer comprising mammalian lung fibroblast cells onto a second side of the porous membrane that is opposite the first side of the porous membrane; and
depositing an epithelial cell layer comprising human lung epithelial cells directly onto the stromal cell layer,
thereby making the lung organoid, wherein cells of the epithelial cell layer of the lung organoid express at least one marker selected from the group consisting of p63+ KRT5+, Foxj1+ Sox17+, Scgb1A1+, Muc5ac+, and CFTR+.