US 12,006,514 B2
Method for the isolation of subpopulations of cardiac progenitor cells and related uses in the medical field
Giulio Pompilio, Milan (IT); Elisa Gambini, Sovico (IT); and Gabriella Spaltro, Truccazzano (IT)
Assigned to OLOKER THERAPEUTICS S.R.L., Bari (IT)
Appl. No. 16/617,429
Filed by CENTRO CARDIOLOGICO MONZINO SPA, Milan (IT); and UNIVERSITA' DEGLI STUDI DI MILANO, Milan (IT)
PCT Filed Jun. 6, 2018, PCT No. PCT/IB2018/054057
§ 371(c)(1), (2) Date Nov. 26, 2019,
PCT Pub. No. WO2018/224983, PCT Pub. Date Dec. 13, 2018.
Claims priority of application No. 102017000062176 (IT), filed on Jun. 7, 2017.
Prior Publication US 2020/0140818 A1, May 7, 2020
Int. Cl. C12N 5/077 (2010.01); A61K 35/34 (2015.01); G01N 1/28 (2006.01); G01N 33/569 (2006.01)
CPC C12N 5/0657 (2013.01) [A61K 35/34 (2013.01); G01N 1/286 (2013.01); G01N 33/56966 (2013.01); C12N 2500/30 (2013.01); C12N 2501/115 (2013.01); C12N 2501/14 (2013.01); G01N 2001/2873 (2013.01)] 17 Claims
 
1. A method for the isolation of human cardiac progenitor cells subpopulations, comprising the following steps:
a) mincing a heart tissue sample containing a population of cardiac cells;
b) 4-times progressive digestion of the cardiac tissue with an enzymatic mixture comprising collagenase and excluding trypsin in a concentration of between 0.1 and 3 mg/ml until a cell suspension is achieved and filtration is achieved through filters of size comprised between 30 μm and 100 μm;
c) further digestion of residual cardiac tissue obtained in step b) for 16 hours with an enzymatic mixture comprising collagenase and excluding trypsin in a concentration of between 0.1 and 3 mg/ml until a cell suspension is achieved and filtration through filters of size comprised between 30 μm and 100 μm;
d) culturing the cell suspension obtained in step b) and c) in a culture medium suitable to propagate the cardiac cells;
e) preliminary expansion of the cell suspension obtained in step d) in a culture medium suitable to expand the cardiac cells in the presence of an enzymatic or non-enzymatic solution;
f) secondary expansion of the cardiac cells obtained in step e) in a culture medium suitable to further expand the cardiac cells in the presence of an enzymatic or non enzymatic non-enzymatic solution;
g) isolating one or more subpopulations of cardiac cells by positive and/or negative selection by the use of monoclonal antibodies directed against one or more surface antigens expressed in the starting population of cardiac progenitor cells.