# US 10,369,173 B2 · Optimized methods for generation of cardiac stem cells from cardiac tissue and their use in cardiac therapy

> Agents: this data is available as JSON from the patentgrep API and MCP server — see https://patentgrep.com/llms.txt

- Number: US 10,369,173 B2
- Document ID: US10369173B2
- Status: Granted
- Kind: B2
- Application number: 15/837,549
- Filed: 2017-12-11
- Published: 2018-06-21
- Granted: 2019-08-06
- Earliest claimed: 2012-06-05
- Assignees: Capricor, Inc.
- Applicants: Capricor, Inc.
- Inventors: Michelle Kreke; Rachel Smith
- Web page: https://patentgrep.com/patent/US10369173B2

## CPC

- A61K 35/34: Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes (vascular smooth muscle A61K35/44)
- A61P 9/00: Drugs for disorders of the cardiovascular system
- A61P 9/10: for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
- C12N 5/0657: {Cardiomyocytes; Heart cells}
- A61K 35/00: Medicinal preparations containing materials or reaction products thereof with undetermined constitution
- C12N 2500/32: Amino acids
- C12N 2501/91: Heparin
- C12N 2509/00: Methods for the dissociation of cells, e.g. specific use of enzymes
- C12N 2523/00: Culture process characterised by temperature
- A61K: PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES (devices or methods specially adapted for bringing pharmaceutical products into particular physical or administering forms A61J3/00; chemical aspects of, or use of materials for deodorisation of air, for disinfection or sterilisation, or for bandages, dressings, absorbent pads or surgical articles A61L; soap compositions C11D)
- A61P: SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
- C12N: MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA (microbiological testing media C12Q1/00)

## 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.

## Claims

### Claim 1

A method for preparing filtered cardiosphere-derived cells (CDCs) suitable for allogeneic cardiac stem cell therapy, comprising:
receiving donor cardiac tissue from a subject;
processing said piece of donor cardiac tissue into a plurality of tissue explants ranging from about 200 μm3 to about 600 μm3, wherein said explants range from about 0.3 to about 0.6 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 cm2 culturing surface area until cells migrate from said explant;
collecting the cells that migrate from the explant;
culturing the collected cells in order to generate CDCs;
harvesting the CDCs; and
filtering said harvested CDCs to remove particles greater than 50 μm in diameter to isolate CDCs less than 50 μm in diameter to facilitate allogeneic cardiac stem cell therapy.

### Claim 2 (depends on 1)

The method of claim 1, wherein the isolated CDCs that are sized less than 50 μm in diameter are suitable for intracoronary delivery and passage into cardiac arterioles having an inner diameter of about 50 μm.

### Claim 3 (depends on 1)

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 μm and through a second filter to remove particles greater than about 50 μm.

### Claim 4 (depends on 3)

The method of claim 3, wherein said first and said second filter comprise a single two-stage filter device.

### Claim 5 (depends on 1)

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.

### Claim 6 (depends on 1)

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.

### Claim 7 (depends on 1)

The method of claim 1, wherein said plurality of explants comprises a plurality of explants obtained from one region of the donor cardiac tissue.

### Claim 8 (depends on 1)

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.

### Claim 9 (depends on 1)

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.

### Claim 10 (depends on 1)

The method of claim 1, further comprising freezing the CDCs suitable for allogeneic cardiac stem cell therapy.

### Claim 11 (depends on 1)

The method of claim 1, wherein the donor cardiac tissue ranges in size from about 1 gram to about 300 grams.

### Claim 12 (depends on 1)

The method of claim 1, wherein said processing yields a plurality of tissue explants that are cuboidal in shape.

### Claim 13 (depends on 1)

The method of claim 1, wherein the donor cardiac tissue is processed within 3 days from removal from said subject.

### Claim 14 (depends on 1)

The method according to claim 1, wherein said CDCs suitable for allogeneic cardiac stem cell therapy comprise less than 1% particles greater than 140 μm.

### Claim 15 (depends on 1)

The method according to claim 1, wherein said CDCs suitable for allogeneic cardiac stem cell therapy comprise essentially 0% particles greater than 150 μm.

### Claim 16 (depends on 1)

The method according to claim 1, wherein said CDCs suitable for allogeneic cardiac stem cell therapy comprise less than 1% particles greater than 50 μm.

### Claim 17 (depends on 1)

The method according to claim 1, further comprising delivering said isolated CDCs to a subject via intracoronary delivery.

### Claim 18

A method for preparing cardiosphere derived cells (CDCs), comprising:
processing cardiac tissue into a plurality of tissue explants;
culturing said explants until cells migrate from said explant;
collecting the cells that migrate from the explant;
culturing the collected cells in order to generate CDCs;
harvesting the CDCs;
filtering said harvested CDCs remove particles greater than about 50 μm.

### Claim 19 (depends on 18)

The method according to claim 18, wherein said cardiac tissue is processed into a plurality of tissue explants ranging from about 0.3 to about 0.6 grams.

### Claim 20 (depends on 18)

The method according to claim 18, wherein said explants are cultured at a density of about 1 explant per 400 to 700 cm2 culturing surface area.