Method of constructing masses of myocardial cells and use of the myocardial cell mass
Inventors
Hattori, Fumiyuki • Fukuda, Keiichi
Assignees
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Publication Number
US-10918671-B2
Publication Date
2021-02-16
Expiration Date
Abstract
The object of the present invention is to improve the post-transplantation engraftment rate of cardiomyocytes that have been purified to such an extent that they are free from non-cardiomyocytes and any components derived from other species.To solve this problem, the present inventors studied the possibility of constructing cell masses from the purified cardiomyocytes. As a result, they revealed that the stated problem could be solved by providing a method of preparing cell masses of cardiomyocytes derived from pluripotent stem cells, characterized in that cell masses of aggregated cells containing cardiomyocytes that had been differentiated and induced from pluripotent stem cells were dispersed to single cells to thereby obtain purified cardiomyocytes, which were then cultured in a culture medium under serum-free conditions so that they were reaggregated.
Core Innovation
The invention relates to treating myocardial infarction in a mammal using pluripotent stem cell-derived cardiomyocytes. It differentiates isolated mammalian pluripotent stem cells into aggregated cell masses comprising cardiomyocytes, disperses the aggregated cell masses to obtain single cardiomyocytes, and purifies the single cardiomyocytes before further processing.
The purified single cardiomyocytes are cultured in a culture medium under serum-free conditions for at least 12 hours, such that the single cardiomyocytes form reaggregated masses of cardiomyocytes. The disclosed approach uses serum-free reaggregation to form spherical cell masses that beat synchronously, and it further describes reaggregation with reaggregation-promoting growth factors while avoiding non-cardiomyocyte mixing.
The invention further encompasses preparing transplantable cardiomyocyte formats from the serum-free reaggregated masses, including transplanting reaggregated masses or transplanting a sheet of reaggregated masses of cardiomyocytes. The disclosed sheet is characterized by thickness, and the invention also describes detecting and rejecting contaminating proliferative non-cardiomyocytes based on cell mass characteristics and mitochondrial dye signal, to improve purity and safety for transplantation.
Claims Coverage
Across the provided material, three independent claims are directed to methods of treating myocardial infarction in a mammal. The inventive coverage centers on a serum-free reaggregation workflow for purified single cardiomyocytes derived from aggregated pluripotent stem cell cardiomyocytes, combined with specific transplantable formats and morphology/thickness constraints.
Serum-free reaggregation of purified single cardiomyocytes for transplantation
Differentiating isolated mammalian pluripotent stem cells into aggregated cell masses comprising cardiomyocytes; dispersing the aggregated cell masses comprising cardiomyocytes such that single cardiomyocytes are obtained; purifying the single cardiomyocytes; culturing the purified single cardiomyocytes in a culture medium under serum-free conditions for at least 12 hours such that the single cardiomyocytes form reaggregated masses of cardiomyocytes; and transplanting the reaggregated masses of cardiomyocytes into a site of myocardial infarction in the mammal such that a symptom of the myocardial infarction is treated.
Transplantation of a sheet of serum-free reaggregated cardiomyocyte masses
Differentiating isolated mammalian pluripotent stem cells into aggregated cell masses comprising cardiomyocytes; dispersing the aggregated cell masses comprising cardiomyocytes such that single cardiomyocytes are obtained; purifying the single cardiomyocytes; culturing the purified single cardiomyocytes in a culture medium under serum-free conditions for at least 12 hours such that the single cardiomyocytes form reaggregated masses of cardiomyocytes; and transplanting a sheet of the reaggregated masses of cardiomyocytes into a site of myocardial infarction in the mammal such that a symptom of the myocardial infarction is treated.
Seeded cardiomyocyte sheet formation from serum-free reaggregated masses
Differentiating isolated mammalian pluripotent stem cells into aggregated cell masses comprising cardiomyocytes; dispersing the aggregated cell masses comprising cardiomyocytes such that single cardiomyocytes are obtained; purifying the single cardiomyocytes; culturing the purified single cardiomyocytes in a culture medium under serum-free conditions for at least 12 hours such that the single cardiomyocytes form reaggregated masses of cardiomyocytes; seeding the reaggregated masses of cardiomyocytes on a surface; maintaining a suspension culture of the seeded reaggregated masses of cardiomyocytes until a sheet of cardiomyocytes having a thickness of 50-300 μm is formed; and transplanting the sheet of cardiomyocytes into a site of myocardial infarction in the mammal such that a symptom of the myocardial infarction is treated.
The claims consistently require differentiation to aggregated cardiomyocyte masses, dispersal to purified single cardiomyocytes, serum-free culturing for at least 12 hours to reaggregate into cardiomyocyte masses, and transplantation to treat myocardial infarction symptoms. Additional claim coverage constrains the transplantable format to a cardiomyocyte sheet and, in one independent claim, defines sheet formation via seeding and suspension culture with a thickness of 50-300 μm.
Stated Advantages
Improves engraftment after transplantation compared with injected dispersed cells, with reported improvement up to ~92% engraftment versus ~0%.
Enables efficient formation of spherical cardiomyocyte cell masses that beat synchronously.
Yields high viability (reported ~90%+).
Supports maturation in host tissue.
Allows detection and rejection of contaminating proliferative non-cardiomyocytes to address purity and safety concerns.
Documented Applications
Treating myocardial infarction in a mammal by transplanting serum-free reaggregated cardiomyocyte masses or cardiomyocyte sheets to treat a symptom of the myocardial infarction.
Providing a transplantation-ready medical device comprising the engineered cell masses or sheets.
Cardiac disease treatment including myocardial infarction in embodiments described with human cardiomyocyte engraftment evidence in immunodeficient mice.
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