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-9109205-B2
Publication Date
2015-08-18
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 preparing cell masses of cardiomyocytes derived from pluripotent stem cells by culturing a purified fraction of single cells of cardiomyocytes derived from pluripotent stem cells in a culture medium under serum-free conditions in suspension culture conditions in a round-bottomed well such that they are aggregated to form cell masses of cardiomyocytes derived from pluripotent stem cells. The purified fraction of single cells of cardiomyocytes is obtained by inducing differentiation of pluripotent stem cells into cardiomyocytes to form embryoid bodies, dispersing the embryoid bodies, and purifying a fraction of single cells of cardiomyocytes derived from pluripotent stem cells.
The invention further provides transplantation of the prepared cardiomyocyte cell masses to cardiac tissue of an individual having cardiac disease such that the cell masses of cardiomyocytes derived from pluripotent stem cells are engrafted. In this treating method, the cardiomyocyte cell masses are prepared by the same serum-free suspension aggregation of purified single-cell cardiomyocytes in a round-bottomed well into cardiomyocyte cell masses, and the purified single-cell fraction is produced via embryoid body formation, dispersing, and purification.
In addition, the invention provides preparing a sheet of cell masses of cardiomyocytes by seeding cardiomyocyte cell masses on the surface of a wall-partitioned, non-cell-adhering vessel without space between cell masses so adjacent cell masses are continuously in contact, followed by suspension culture maintained until adjacent cell masses conjugate together to have a desired thickness of 50-300 5m. The cardiomyocyte cell masses used for the sheet are obtained by the serum-free suspension aggregation process of purified single cardiomyocytes derived from pluripotent stem cell differentiation through embryoid bodies, dispersing, and purifying.
Claims Coverage
The document includes four independent claims, covering preparing aggregated cardiomyocyte cell masses, treating cardiac disease by transplanting engraftable cardiomyocyte cell masses, preparing a sheet of cardiomyocyte cell masses with a specified thickness, and a medical device for transplantation to cardiac tissue using those prepared cardiomyocyte cell masses. Across the independent claims, the main inventive theme is serum-free suspension aggregation of purified single cardiomyocytes derived from pluripotent stem cells differentiated via embryoid bodies, with additional formats and a sheet-thickness specification.
Serum-free suspension aggregation of purified single-cell cardiomyocytes in a round-bottomed well
Culturing a purified fraction of single cells of cardiomyocytes derived from pluripotent stem cells in a culture medium under serum-free conditions in suspension culture conditions in a round-bottomed well so that they are aggregated to form cell masses of cardiomyocytes derived from pluripotent stem cells.
Embryoid body differentiation, dispersing, and purification to obtain the purified single-cell fraction
Obtaining the purified fraction of single cells of cardiomyocytes by inducing differentiation of pluripotent stem cells into cardiomyocytes resulting to form embryoid bodies, dispersing the embryoid bodies and purifying a fraction of single cells of cardiomyocytes derived from pluripotent stem cells.
Transplantation to cardiac tissue for engraftment to treat cardiac disease
Transplanting cell masses of cardiomyocytes derived from pluripotent stem cells to the cardiac tissue of an individual such that they are engrafted, wherein the cell masses are obtained by serum-free suspension culturing of a purified fraction of single cardiomyocytes in a round-bottomed well to form cell masses, with the purified fraction obtained by embryoid body differentiation, dispersing, and purifying.
Sheet formation by conjugating adjacent cell masses on a wall-partitioned non-cell-adhering vessel to a desired thickness
Seeding cell masses of cardiomyocytes derived from pluripotent stem cells on the surface of a wall-partitioned, non-cell-adhering vessel, with no space between cell masses so that adjacent cell masses will be continuously in contact, followed by suspension culture maintained until the cell masses are conjugated together to have a desired thickness of 50-300 5m.
Medical device comprising transplantable cardiomyocyte cell masses for cardiac tissue
Providing a medical device for transplantation to the cardiac tissue of an individual having a cardiac disease, the medical device comprising cell masses of cardiomyocytes derived from pluripotent stem cell, wherein the cell masses are obtained by serum-free suspension culture of a purified fraction of single cardiomyocytes aggregated in a round-bottomed well and wherein the purified single-cell fraction is obtained by inducing differentiation of pluripotent stem cells into cardiomyocytes resulting to form embryoid bodies, dispersing the embryoid bodies and purifying a fraction of single cells of cardiomyocytes derived from pluripotent stem cells.
Across the independent claims, the patent focuses on preparing cardiomyocyte cell masses by aggregating a purified fraction of single pluripotent-stem-cell-derived cardiomyocytes under serum-free suspension conditions in round-bottomed wells, where the purified single-cell fraction is produced via embryoid body differentiation, dispersing, and purification. Claim coverage extends this prepared material to treating cardiac disease by transplanting engraftable cardiomyocyte cell masses into cardiac tissue, including a sheet format formed on a wall-partitioned non-cell-adhering vessel to a desired thickness of 50-300 5m, and a corresponding medical device for transplantation.
Stated Advantages
Dramatically increased engraftment when transplanting reaggregated purified cardiomyocyte cell masses compared with injection of dispersed single cells.
Efficient reaggregation of embryonic stem cell-derived purified cardiomyocytes under serum-free conditions.
Improved cell mass formation, viability, and survival when insulin (ITS) is added.
Documented Applications
Treating cardiac disease by transplanting cell masses of cardiomyocytes derived from pluripotent stem cells into cardiac tissue of an individual such that the cell masses are engrafted.
Transplanting a medical device comprising cardiomyocyte cell masses derived from pluripotent stem cells to cardiac tissue of an individual having cardiac disease.
Preparing a sheet of cell masses of cardiomyocytes with a desired thickness of 50-300 5m by conjugating adjacent cell masses formed from serum-free suspension aggregation.
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