Systems and method for engineering muscle tissue

Patent metadata

Field	Value
Publication number	US-9994812-B2
Application number	US-201314390490-A
Country	US
Kind code	B2
Filing date	Mar 15, 2013
Priority date	Apr 4, 2012
Publication date	Jun 12, 2018
Grant date	Jun 12, 2018

Abstract

The present invention generally relates to the field of cell growth and tissue engineering, in particular, tissue engineered compositions comprising a nanotextured substrate which is structurally configured for growth of cells in an anatomically correct adult phenotype in vitro. In particular, described herein are nanotextured substrates which are structurally configured for the anisotropic organization, maturation, and growth of in vitro-differentiated muscle cells, such as cardiomyocytes, and methods for the production and use thereof in varying sizes, nanotextures and substrate rigidities. In vitro-differentiated cardiomyocytes grown on the nanotextured substrates described herein are better-differentiated and more closely mimic adult cardiac tissue than the same cells grown on a non-textured substrate of the same composition. The nanotextured substrate/cell constructs provide a platform for screening to predict the effect of test agents or drugs on, for example, human cardiac tissue, including patient-derived tissue, or for the identification of agents that effect various cardiac functional parameters.

First claim

The invention claimed is:

1. A nanotextured platform composition comprising a polymer substrate comprising:
a. a nanotextured array of parallel grooves and ridges that organizes cultured human cardiomyocytes in an anisotropic manner, the grooves having a width of 200-1000 nm, and said ridges, between said grooves, having a width of 200-1000 nm, said nanotextured array providing nanotopographic cues guiding maturation of cells cultured on said nanotextured array; and
b. in vitro-differentiated human cardiomyocytes cultured on said nanotextured array, said in vitro differentiated human cardiomyocytes being organized in an anisotropic manner in response to said nanotopographic cues wherein:
(i) said in vitro-differentiated human cardiomyocytes are differentiated from human embryonic stem cells (ESCs) or from induced pluripotent stem cells (iPSCs) derived from an adult somatic cell of either a healthy individual or an individual diagnosed with a cardiac disease;
(ii) said in vitro-differentiated human cardiomyocytes have at least one of the following characteristics of maturity:
(a) express at least one marker from the group consisting of: Nkx2.5, GATA4, connexin-43, myosin heavy chain and cTNT, in a manner more similar to adult cardiomyocytes than in vitro differentiated cardiomyocytes cultured on a polymer substrate of the same composition but substantially lacking said nanotextured array;
(b) isoform switching of ssTn1 to ctTn1 or N2Ba to N2B by at least 10%, as measured by Western Blot or qRT-PCR, to a profile more similar to adult cardiomyocytes than in vitro differentiated cardiomyocytes cultured on a polymer of the same composition but substantially lacking said nanotextured array; and

(iii) said in vitro-differentiated human cardiomyocytes maintain the at least one characteristic noted in (ii) above for 10 days or more in culture.

2. The nanotextured platform of claim 1 , further comprising an agent that promotes the differentiation of the human cardiomyocytes to a more mature phenotype.

3. The nanotextured platform of claim 1 , wherein said grooves have a depth of 50-1000 nm.

4. The nanotextured platform of claim 1 , wherein said grooves have a width of 200-800 nm, and said ridges, between said grooves, have a width of 200-800 nm.

5. The nanotextured platform of claim 1 , wherein said cardiomyocytes form a monolayer on said substrate with anisotropic and polarized cell arrangement in the direction of the nanotextures.

6. The nanotextured platform of claim 1 , wherein said array of parallel grooves and ridges has a precision of texture of at least 90% fidelity, as determined by atomic force microscopy or electron microscopy.

7. The nanotextured platform of claim 1 , wherein said array of parallel grooves and ridges is generated using a process selected from the group consisting of capillary force lithography, nanoindentation, e-beam lithography, and electrospinning.

8. The nanotextured platform of claim 1 , wherein said array of parallel grooves and ridges is formed by capillary force lithography.

9. The nanotextured platform of claim 1 , wherein the nanotextured platform is coated with or wherein the substrate comprises within its polymer matrix, either biocompatible extracellular matrix polypeptides, engineered matrix polypeptides, or engineered polypeptides.

10. The nanotextured platform of claim 1 , further comprising a coating with a materials selected from the group consisting of charcoal, graphene, graphene oxide, reduced graphene oxide, nanotubes, and gold, said coating effecting one or more functional parameters of the substrate selected from: adsorption of proteins to surface, electrical conductivity or other physico-chemical property in a manner that influences the phenotype of said cardiac cells.

11. The nanotextured platform of claim 1 , wherein said substrate comprises a polymer hydrogel comprising, within the matrix of said polymer substrate, a biocompatible extracellular matrix protein, a synthetic or engineered matrix polypeptide, or engineered polypeptides.

12. The nanotextured platform of claim 1 , wherein said polymer substrate comprises a UV curable hydrogel polymer, a thermosensitive hydrogel polymer or a polymer produced by solvent evaporation.

13. The nanotextured platform of claim 1 , configured as a multi-well plate.

14. The nanotextured platform of claim 13 , wherein the multi-well plate is a 24, 48 or 96 well plate.

Assignees

• Univ Washington Through Its Center For Commercialization

Inventors

• Kim Deok-Ho
• Laflamme Michael
• Murry Charles
• Gupta Kshitiz
• Yoo Hyok
• Jiao Alex

Classifications

• C12N5/0657

  Cardiomyocytes; Heart cells · CPC title

• A61L27/3826

  Muscle cells, e.g. smooth muscle cells · CPC title

• C12M21/08

  for producing artificial tissue or for ex-vivo cultivation of tissue (prostheses A61F2/00, grafts A61L27/00) · CPC title

• C12N2535/00

  Supports or coatings for cell culture characterised by topography · CPC title

• C12N2503/02

  Drug screening · CPC title