	US 12,139,722 B2
	Methods for generating and using organoids and tissue therein
Timothy D. O'Brien, Cokato, MN (US); Beth Lindborg, St. Paul, MN (US); and Amanda Vegoe, Apple Valley, MN (US)
Assigned to REGENTS OF THE UNIVERSITY OF MINNESOTA, Minneapolis, MN (US)
Filed by REGENTS OF THE UNIVERSITY OF MINNESOTA, Minneapolis, MN (US)
Filed on Jun. 10, 2021, as Appl. No. 17/344,064.
Application 17/344,064 is a continuation of application No. PCT/US2019/067150, filed on Dec. 18, 2019.
Claims priority of provisional application 62/781,858, filed on Dec. 19, 2018.
Prior Publication US 2021/0301251 A1, Sep. 30, 2021
Int. Cl. C12N 5/00 (2006.01); C12N 5/077 (2010.01)

CPC C12N 5/0062 (2013.01) [C12N 5/0655 (2013.01); C12N 2500/02 (2013.01); C12N 2501/15 (2013.01); C12N 2501/155 (2013.01); C12N 2501/19 (2013.01); C12N 2501/905 (2013.01)]

15 Claims

1. A method comprising:

introducing an induced pluripotent stem cell (iPSC) into a cell culture medium lacking a three-dimensional matrix, the culture medium comprising:

a chemically defined maintenance cell culture medium, and

hyaluronic acid in an amount effective to induce the iPSC to differentiate; and

transferring the iPSC and culture medium to a non-microfluidic bioreactor, the bioreactor comprising a non-adherent culture without the use of a three-dimensional matrix;

culturing the iPSC in the bioreactor for at least 1 week at a temperature from 32° C. to 40° C.; and

producing a three-dimensional multi-tissue organoid comprising cartilage, bone, fibrous connective tissue, brain tissue, or epithelial tissue, or a combination thereof.