Claims for Patent: 9,884,076

Summary for Patent: 9,884,076

Title:	Optimized methods for generation of cardiac stem cells from cardiac tissue and their use in cardiac therapy
Abstract:	The present disclosure relates generally to methods for the increased processing of tissue for the generation of cardiac stem cells, wherein the stem cells are suitable for use in cardiac stem cell therapy. In particular, several embodiments relate to the processing of allogeneic donor cardiac tissue for the generation of multiple patient doses of cardiac stem cells.
Inventor(s):	Kreke; Michelle (Culver City, CA), Smith; Rachel (Sherman Oaks, CA)
Assignee:	Capricor, Inc. (Los Angeles, CA)
Application Number:	14/403,078
Patent Claims:	1. A method for preparing filtered cardiosphere-derived cells (CDCs) for allogeneic cardiac stem cell therapy, comprising the steps of: processing donor cardiac tissue into a plurality of tissue explants ranging from about 100 .mu.m.sup.3 to about 800 .mu.m.sup.3, wherein each of said explants range from about 0.1 to about 0.9 grams; enzymatically digesting said explants for a time ranging from about 30 seconds to about 5 minutes; culturing said explants at a density of about 1 explant per 400 to 700 cm.sup.2 culturing surface area until cells migrate from said explant; collecting the cells that migrate from the explant; culturing the collected cells to generate cardiospheres (CSps); culturing the generated CSps by seeding on a fibronectin-coated dish, plate, or vessel to generate CDCs as a monolayer culture; harvesting the generated CDCs; and filtering said harvested CDCs to remove particles greater than 50 .mu.m in diameter, thereby generating filtered CDCs for allogeneic cardiac stem cell therapy. 2. The method of claim 1, wherein the donor cardiac tissue ranges in size from about 1 gram to about 300 grams, wherein the enzymatic digestion comprises digestion of the explants with trypsin and/or collagenase, wherein the cells that migrate from the explant are collected when confluent, and cryopreserving at least a portion of the harvested CDCs. 3. The method of claim 1, wherein the filtered CDCs are sized less than 50 .mu.m in diameter and are suitable for intracoronary delivery and passage into cardiac arterioles having an inner diameter of about 50 .mu.m. 4. The method of claim 1, wherein said filtering comprises filtering said harvested CDCs through a first filter to remove particles greater in size than about 140 .mu.m and through a second filter to remove particles greater than about 50 .mu.m. 5. The method of claim 4, wherein said first and said second filter comprise a single two-stage filter device. 6. The method of claim 1, wherein said donor cardiac tissue is optionally cryopreserved for a period of time ranging from about 1 to about 90 days prior to said processing. 7. The method of claim 1, wherein said processing further comprises transfer of said explant from a dissection vessel to a culture vessel by flooding the explant with culture media rather than hand-placing the explant in the culture vessel, wherein said transfer by flooding reduces risk of contamination and reduces perturbation of said explant. 8. The method of claim 1, wherein said plurality of explants comprises a plurality of explants obtained from one region of the donor cardiac tissue. 9. The method of claim 1, wherein said plurality of explants comprises a plurality of explants obtained from more than one region of the donor cardiac tissue. 10. The method of claim 1, wherein said culturing further comprises addition of one or more of heparin and L-glutamine to a culture media in order to reduce clumping of cells migrating from said explant. 11. A method for preparing filtered cardiosphere-derived cells (CDCs) suitable for allogeneic cardiac stem cell therapy, comprising: processing donor cardiac tissue into a plurality of tissue explants, each explant being from about 100 .mu.m.sup.3 to about 800 .mu.m.sup.3; enzymatically digesting said explants; culturing, after said digestion, said explants at a density of about 1 explant per 400 to 700 cm.sup.2 culturing surface area until cells migrate from said explant; collecting the cells that migrate from the explant; culturing the collected cells to generate cardiospheres (CSps); culturing the generated CSps to generate CDCs as a monolayer culture; harvesting the CDCs; and filtering said harvested CDCs through a first filter to remove particles greater in size than about 140 .mu.m and through a second filter to remove particles greater than about 50 .mu.m, thereby generating filtered CDCs suitable for allogeneic cardiac stem cell therapy. 12. The method of claim 11, further comprising freezing the CDCs for allogeneic cardiac stem cell therapy. 13. The method of claim 11, wherein the donor cardiac tissue ranges in size from about 1 gram to about 300 grams, and wherein said step of culturing the generated CSps to generate CDCs as a monolayer culture comprises seeding CSps on a fibronectin-coated dish, plate, or vessel. 14. The method of claim 11, wherein said donor cardiac tissue is processed to yields explants that are cuboidal in shape. 15. The method of claim 11, wherein the donor cardiac tissue is processed within 3 days from removal from said subject. 16. The method of claim 11, further comprising storing said received donor cardiac tissue for about three to about six days prior to said processing. 17. A method for preparing cardiosphere derived cells (CDCs) for allogeneic cardiac stem cell therapy, comprising: dividing donor cardiac tissue according to the region of the heart from which the donor tissue originated; processing said donor cardiac tissue into a plurality of cuboidal tissue explants, each explant weighing between about 0.1 to about 0.9 grams; culturing said explants until cells migrate from said explant; collecting the cells that migrate from the explant; culturing the collected cells to generate cardiospheres (CSps); culturing the generated CSps by seeding the CSps on a dish, plate, or vessel that generates CDCs as a monolayer culture; harvesting the CDCs; filtering said harvested CDCs remove particles greater than about 50 .mu.m, thereby generating CDCs for allogeneic cardiac stem cell therapy. 18. The method of claim 17, wherein the explants are cultured such that each explant is allotted 400 to 700 cm.sup.2 culturing surface area. 19. The method of claim 17, wherein said culturing of said explants comprises culturing explants originating from different regions of the heart. 20. The method of claim 17, wherein said culturing of said explants comprises culturing explants originating from the same region of the heart.

Details for Patent 9,884,076

Applicant	Tradename	Biologic Ingredient	Dosage Form	BLA	Approval Date	Patent No.	Expiredate
Smith & Nephew, Inc.	SANTYL	collagenase	Ointment	101995	06/04/1965		2032-06-05