Cellular seeding and co-culture of a three dimensional fibroblast construct

• US 9,976,123 B2
• Filed: 05/21/2015
• Issued: 05/22/2018
• Est. Priority Date: 04/09/2009
• Status: Active Grant

• Current Assignee: US Department of Veterans Affairs (Government of the United States of America), Arizona Board of Regents (University of Arizona)
• Original Assignee: US Department of Veterans Affairs (Government of the United States of America), Arizona Board of Regents (University of Arizona)
• Inventors: Lancaster, Jordan, Goldman, Steve
• Primary Examiner(s): JOHNSON, KARA D
• Application Number:
  US14/718,309
• Publication Number:
  US 20150328367A1
• Time in Patent Office: 1,097 Days
• CPC Class Codes: A61K 35/33 Fibroblasts
• CPC Class Codes: A61K 35/34 Muscles; Smooth muscle cells; Heart; Cardiac stem cells; Myoblasts; Myocytes; Cardiomyocytes vascular smooth muscle A61K35/44
• CPC Class Codes: A61K 35/44 Vessels; Vascular smooth muscle cells; Endothelial cells; Endothelial progenitor cells
• CPC Class Codes: A61K 45/06 Mixtures of active ingredients without chemical characterisation, e.g. antiphlogistics and cardiaca
• CPC Class Codes: A61L 2300/64 Animal cells
• CPC Class Codes: A61L 2430/20 for reconstruction of the heart, e.g. heart valves
• CPC Class Codes: A61L 27/3604 characterised by the human or animal origin of the biological material, e.g. hair, fascia, fish scales, silk, shellac, pericardium, pleura, renal tissue, amniotic membrane, parenchymal tissue, fetal tissue, muscle tissue, fat tissue, enamel
• CPC Class Codes: A61L 27/367 Muscle tissue, e.g. sphincter
• CPC Class Codes: A61L 27/3826 Muscle cells, e.g. smooth muscle cells
• CPC Class Codes: A61P 1/08 for nausea, cinetosis or vertigo; Antiemetics
• CPC Class Codes: A61P 1/16 for liver or gallbladder disorders, e.g. hepatoprotective agents, cholagogues, litholytics
• CPC Class Codes: A61P 11/00 Drugs for disorders of the respiratory system
• CPC Class Codes: A61P 13/02 of urine or of the urinary tract, e.g. urine acidifiers
• CPC Class Codes: A61P 9/00 Drugs for disorders of the cardiovascular system
• CPC Class Codes: A61P 9/04 Inotropic agents, i.e. stimulants of cardiac contraction; Drugs for heart failure
• CPC Class Codes: A61P 9/10 for treating ischaemic or atherosclerotic diseases, e.g. antianginal drugs, coronary vasodilators, drugs for myocardial infarction, retinopathy, cerebrovascula insufficiency, renal arteriosclerosis
• CPC Class Codes: C12N 2502/1323 Adult fibroblasts
• CPC Class Codes: C12N 2502/28 Vascular endothelial cells
• CPC Class Codes: C12N 5/0062 General methods for three-dimensional culture
• CPC Class Codes: C12N 5/0657 Cardiomyocytes; Heart cells

Abstract

The present invention provides methods for cellular seeding onto three-dimensional fibroblast constructs, three-dimensional fibroblast constructs seeded with muscle cells, and uses therefore.

Claims

1. A method for treating a disorder characterized by a lack of functioning cardiomyocytes, comprising contacting the heart of a subject suffering from such a disorder with an amount effective to treat the disorder of a construct comprising cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to a three dimensional fibroblast construct (3DFC), wherein the construct is capable of spontaneous synchronized contractions across the 3DFC;

and wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof are seeded on the construct at a density of between 0.5×

10⁶ cells/cm² and 5×

10⁶ cells/cm² and/or the cardiomyocytes, cardiac stem cells, or progenitors thereof are present in a ratio of between about 1;

10 and about 10;

1 with fibroblasts on the 3DFC;

wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof adhered as a 1-2 cell thick layer over a layer of fibroblasts on

(a) the top surface of the 3DFC, wherein the bottom surface of the 3DFC does not have cardiomvocvtes, cardiac stem cells, or progenitors thereof adhered to it,

(b) the bottom surface of the 3DFC, wherein the top surface of the 3DFC does not have cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to it;

or

(c) the top and the bottom surface of the 3DFC.

2. The method of claim 1, wherein the disorder is selected from the group consisting of chronic heart failure (CHF), ischemia without heart failure, cardiomyopathy, dilated cardiomyopathy (DCM), cardiac arrest, congestive heart failure, stable angina, unstable angina, myocardial infarction, coronary artery disease, valvular heart disease, ischemic heart disease, reduced ejection fraction, reduced myocardial perfusion, maladaptive cardiac remodeling, left ventricle remodeling, reduced left ventricle function, left heart failure, right heart failure, backward heart failure, forward heart failure, systolic dysfunction, diastolic dysfunction, systemic vascular resistance, low-output heart failure, high-output heart failure, dyspnea on exertion, dyspnea at rest, orthopnea, tachypnea, paroxysmal nocturnal dyspnea, dizziness, confusion, cool extremities at rest, exercise intolerance, easy fatigueability, peripheral edema, nocturia, ascites, hepatomegaly, pulmonary edema, cyanosis, laterally displaced apex beat, gallop rhythm, heart murmurs, parasternal heave, and pleural effusion.

3. The method of claim 2, where the disorder comprises CHF.

4. The method of claim 2, where the disorder comprises DCM.

5. The method of claim 2, where the disorder comprises left heart failure.

6. The method of claim 2, where the disorder comprises reduced left ventricular function.

7. The method of claim 2, where the disorder comprises right heart failure.

8. The method of claim 1, wherein the construct is attached to the epicardium.

9. The method of claim 8, wherein the construct is non-contracting at the time of contacting with the subject'"'"'s heart.

10. The method of claim 1, wherein the contacting comprises contacting the construct to the subject'"'"'s left ventricle.

11. The method of claim 1, further comprising contacting the subject'"'"'s heart with one or more of thymosin beta-4 (TB4), akt murine thyoma viral oncogene homolog (AKT1), stroma cell-derived factor-1 alpha (SDF-1), and hepatocyte growth factor (HGF).

12. The method of claim 1, wherein the treating comprises one or more of improving left ventricular function, fall in end diastolic pressure (EDP), improving myocardial perfusion, repopulating of the heart'"'"'s anterior wall with cardiomyocytes, and reversing maladaptive left ventricle remodeling in CHF subjects.

13. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are present on the construct in a ratio between about 1:

10 and about 10;

1 fibroblasts.

14. The method of claim 1, herein the cardiomyocytes or cardiac stem cells are seeded on the construct at a density of between 1.3×

10⁶ cells/cm² and 5×

10⁶ cells/cm².

15. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are seeded on the construct at a density of between 1.5×

10⁶ cells/cm² and 3×

10⁶ cells/cm².

16. The method of claim 1, wherein the cardiomyocytes or cardiac stem cells are of human origin.

17. The method of claim 1, where the disorder comprises reduced ventricular function.