	US 12,435,314 B2
	Method for cultivating stem cells in vitro
Andreas Marg, Berlin (DE); and Simone Spuler, Berlin (DE)
Assigned to Simone Spuler, Berlin (DE)
Filed by Simone Spuler, Berlin (DE)
Filed on Aug. 15, 2022, as Appl. No. 17/888,245.
Application 17/888,245 is a division of application No. 15/506,726, granted, now 11,414,647, previously published as PCT/EP2015/069442, filed on Aug. 25, 2015.
Claims priority of application No. 10 2014 216 872.4 (DE), filed on Aug. 25, 2014.
Prior Publication US 2022/0396770 A1, Dec. 15, 2022
Int. Cl. C12N 5/077 (2010.01); A61K 35/34 (2015.01)

CPC C12N 5/0659 (2013.01) [A61K 35/34 (2013.01); C12N 2500/02 (2013.01); C12N 2523/00 (2013.01)]

12 Claims

1. A method for generating genetically modified human muscle satellite cells, the method comprising the steps of:

obtaining a human muscle biopsy specimen from a living subject;

mechanically isolating, without enzymatic treatment, muscle fiber fragments from the biopsy specimen to provide isolated muscle fiber fragments comprising fiber fragments of a length of 2 mm to 3 mm;

incubating the isolated muscle fiber fragments at about 4° C. to about 8° C. for a period of 5 days to 7 days;

immediately after incubation, culturing the isolated muscle fiber fragments at about 37° C. under conditions sufficient for satellite cells present in the isolated muscle fiber fragments to form cell colonies that outgrow from the muscle fiber fragments; and

collecting the satellite cells from the cell colonies, wherein 70% to 100% of viable cells collected from the cell colonies are satellite cells, and

inserting genetic information into the genome of the satellite cells or replacing defect genetic information with desired genetic information.