Method for the isolation of subpopulations of cardiac progenitor cells and related uses in the medical field
Inventors
POMPILIO, Giulio • GAMBINI, Elisa • SPALTRO, Gabriella
Assignees
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Publication Number
US-12006514-B2
Publication Date
2024-06-11
Expiration Date
Abstract
The present invention relates to a method for the isolation of subpopulations of cardiac progenitor cells from a heart tissue sample, the population thus obtained and the related uses in the medical field for the cell therapy or cardiac cell and/or tissue transplantation field.
Core Innovation
The invention relates to a method for the isolation of human cardiac progenitor cell subpopulations from a heart tissue sample containing a population of cardiac cells. The method includes 4-times progressive digestion of the cardiac tissue using an enzymatic mixture comprising collagenase and excluding trypsin, followed by filtration through filters having a size comprised between 30 μm and 100 μm. Further digestion of residual cardiac tissue for 16 hours using an enzymatic mixture comprising collagenase and excluding trypsin is followed by filtration through filters of the same size range.
The isolated cell suspension is cultured in a culture medium suitable to propagate the cardiac cells, followed by preliminary expansion and secondary expansion in culture media suitable to expand the cardiac cells in the presence of an enzymatic or non-enzymatic solution. The process is followed by isolating one or more cardiac cell subpopulations using positive and/or negative selection with monoclonal antibodies directed against one or more surface antigens expressed in the starting population of cardiac progenitor cells.
The monoclonal antibodies include preferred targets such as anti-CD117 (c-kit) and/or anti-CD90 (Thy1), and the antigen targets may be selected from a Table 1 list. In addition to selection by monoclonal antibodies against surface antigens, the method can use immunomagnetic sorting for positive and/or negative selection. The disclosed approach is presented as a GMP-compatible production method for preparing cardiac progenitor cell subpopulations for cell therapy or transplantation, with stated maintenance of antigen expression after cryopreservation.
Claims Coverage
The document provides one independent claim. The independent claim contains the main inventive sequence: staged collagenase digestion excluding trypsin, size filtration, propagation and staged expansion, and final positive and/or negative isolation of cardiac progenitor cell subpopulations using monoclonal antibodies against surface antigens.
Staged collagenase digestion excluding trypsin with size filtration
Mincing a heart tissue sample containing a population of cardiac cells; 4-times progressive digestion with an enzymatic mixture comprising collagenase and excluding trypsin until a cell suspension is achieved, followed by filtration through filters of size comprised between 30 μm and 100 μm; further digestion of residual cardiac tissue for 16 hours with an enzymatic mixture comprising collagenase and excluding trypsin until a cell suspension is achieved, followed by filtration through filters of size comprised between 30 μm and 100 μm.
Propagation and staged expansion of the isolated cell suspension
Culturing the cell suspension obtained from digestion/filtration in a culture medium suitable to propagate the cardiac cells; preliminary expansion in a culture medium suitable to expand the cardiac cells in the presence of an enzymatic or non-enzymatic solution; secondary expansion in a culture medium suitable to further expand the cardiac cells in the presence of an enzymatic or non-enzymatic solution.
Positive and/or negative selection of subpopulations using monoclonal antibodies against surface antigens
Isolating one or more subpopulations of cardiac cells by positive and/or negative selection using monoclonal antibodies directed against one or more surface antigens expressed in the starting population of cardiac progenitor cells.
Immunomagnetic separation for antibody-based selection
Using immunomagnetic separation in the selection step with the monoclonal antibodies directed against surface antigens expressed in the starting population of cardiac progenitor cells.
Selection using anti-CD117 and/or anti-CD90 monoclonal antibodies
Using monoclonal antibodies directed to anti-CD117 and/or anti-CD90 as the targets for isolating human cardiac progenitor cell subpopulations.
Across the independent claim and its refinements, the core coverage centers on staged collagenase digestion excluding trypsin with size filtration, propagation plus preliminary and secondary expansion, and final isolation of human cardiac progenitor cell subpopulations by positive and/or negative selection using monoclonal antibodies against surface antigens. The refinements further specify immunomagnetic separation and antibody targeting such as anti-CD117 and/or anti-CD90.
Stated Advantages
Scalability from small biopsy samples for GMP-compatible production
Versatility for selecting rare/intermediate/low-antigen-expression cardiac progenitor cell subpopulations
Antigen expression is maintained after cryopreservation
Reduced preparation time versus prior methods
Enhanced angiogenic differentiation, including tubule formation and endothelial marker expression
Increased cytokine/growth factor secretion, including enrichment of IL-6 and IL-8 and other factors listed
Cardioprotective and anti-inflammatory effects in co-culture models, including reduced TNF-α
Reduced collagen/myofibroblast differentiation under TGF-β1 challenge
Documented Applications
Cell and/or tissue transplantation for cardiovascular diseases
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