Cardiosphere-derived cell sheet and methods of making and using the same
A method of making a cell sheet comprising secondary spheroids, including (a) obtaining cardiosphere-derived cells; (b) cultivating the cardiosphere-derived cells for a first period of time in a first media comprising at least one of an ascorbic acid and an analog thereof, to form secondary spheroids; (c) transferring an amount of the spheroids formed in step (b) into a mold; (d) culturing the secondary spheroids for a second period of time in a second media comprising at least one of the ascorbic acid and an analog thereof, wherein the at least one of the ascorbic acid and an analog thereof is present in an amount effective to promote a formation of an extracellular matrix; and (e) culturing a product obtained in step (d) for a third period of time, in the absence of the at least one of the ascorbic acid and an analog thereof.
1. A method of making a cell sheet, comprising:
obtaining a plurality of cardiosphere-derived cells;
cultivating the cardiosphere-derived cells for a first period of time in a first medium comprising at least one of an ascorbic acid and an ascorbic acid analog thereof, to form a plurality of secondary spheroids, wherein the secondary spheroids have a diameter which is greater than 600 μm and not larger than 1450 μm;
transferring an amount of the secondary spheroids into a mold;
culturing the amount of the secondary spheroids in the mold, to obtain secondary spheroids fused with an extracellular matrix, for a second period of time in a second medium comprising the at least one of the ascorbic acid and an ascorbic acid analog thereof, wherein the at least one of the ascorbic acid and an ascorbic acid analog thereof in the second medium is present in an amount effective to promote a formation of the extracellular matrix to which the secondary spheroids fuse; and
culturing the secondary spheroids fused with the extracellular matrix for a third period of time to obtain a cell sheet, in a medium that does not contain ascorbic acid and an ascorbic acid analog thereof,
wherein the first medium and the second medium include a basic fibroblast growth factor in an amount effective to promote the growth of the cardiosphere-derived cells or the secondary spheroids, and wherein the ascorbic acid analog includes at least one of ascorbic acid 2-phosphate, an ascorbic acid 2-phosphate salt thereof, a hydrate of the ascorbic acid 2-phosphate salt, ascorbic acid 3-phosphate, an ascorbic acid 3-phosphate salt thereof, and a hydrate of the ascorbic acid 3-phosphate salt.
2. The method of claim 1 , wherein the first period of time is about 24 hours.
3. The method of claim 1 , wherein the second period of time is about 3 days.
4. The method of claim 1 , wherein the third period of time is about 4 days.
5. The method of claim 1 , wherein a total of the second period of time and the third period of time is about 7 days.
6. The method of claim 1 , wherein the cardiosphere-derived cells obtained have a concentration of between 6.0×10 4 to 2.0×10 5 cells/100 μL.
7. The method of claim 1 , wherein the mold comprises:
a top layer having a shape of a flat sheet comprising at least one aperture;
a base; and
a membrane positioned between the top layer and the base such that the membrane covers the at least one aperture, and
the amount of the secondary spheroids is cultured on the membrane within the at least one aperture.
8. The method of claim 7 , wherein at least one of the top layer and the membrane comprises a non-collagen coated PTFE.
9. The method of claim 7 , wherein at least one of the top layer and the membrane has a pore size of 4 μm to 10 μm.
10. The method of claim 7 , wherein:
the membrane includes pores positioned below the at least one aperture, and
the culturing of the amount of the secondary spheroids includes circulating the second medium in the mold, via diffusion of the second medium through the pores.
11. The method of claim 7 , wherein the top layer comprises four apertures, and each aperture of the four apertures is about 4×4 mm.
12. The method of claim 1 , wherein the first medium includes at least one of Mg salt of ascorbic acid 2-phosphate, Mg hydrate of ascorbic acid 2-phosphate, Mg salt of ascorbic acid 3-phosphate, and Mg hydrate of ascorbic acid 3-phosphate.