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

• US 9,051,550 B2
• Filed: 04/09/2010
• Issued: 06/09/2015
• 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: Arizona Board of Regents (University of Arizona)
• Inventors: Lancaster, Jordan, Goldman, Steve
• Primary Examiner(s): Weber, Jon P
• Assistant Examiner(s): JOHNSON, KARA D
• Application Number:
  US13/260,610
• Publication Number:
  US 20120128637A1
• Time in Patent Office: 1,887 Days
• Field of Search: 424/93.7
• US Class Current: 1/1
• 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 construct comprising 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 a three dimensional fibroblast construct (3DFC), wherein the bottom surface of the 3DFC does not have cardiomyocytes, cardiac stem cells, or progenitors thereof adhered to it,

(b) the bottom surface of the three dimensional fibroblast construct (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;

wherein the cells seeded on the 3DFC surface are capable of initiating spontaneous contractions across the full 3DFC, and wherein the entire construct is capable of synchronized contraction; and

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

10⁶ cells/cm² and 5×

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

10 and about 10;

1 with fibroblasts on the 3DFC.

2. The construct of claim 1, wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof are seeded on the top and/or bottom surface at a density of between 1.3×

10⁶ cells/cm² and 5×

10⁶ cells/cm².

3. The construct of claim 1, wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof are seeded on the top and/or bottom surface at a density of between 1.5×

10⁶ cells/cm² and 3×

10⁶ cells/cm².

4. The construct of claim 1, wherein the cardiomyocytes or cardiac stem cells, or progenitors thereof are of human origin.

5. The construct of claim 1, wherein the cardiomyocytes or cardiac stem cells, or progenitors thereof are engineered to express one or more of thymosin beta-4 (TB4), akt murine thymoma viral oncogene homolog (AKT1), stroma cell-derived factor-1 alpha (SDF-1), and hepatocyte growth factor (HGF).

6. The construct of claim 1, wherein the cardiomyocytes, cardiac stem cells, or progenitors thereof are present on the top and/or bottom surface in a ratio of between about 1:

5 and about 5;

1 with fibroblasts on the 3DFC.