Engineered cell growth on polymeric films and biotechnological application thereof

Patent metadata

Field	Value
Publication number	US-9383350-B2
Application number	US-201313922432-A
Country	US
Kind code	B2
Filing date	Jun 20, 2013
Priority date	Feb 3, 2006
Publication date	Jul 5, 2016
Grant date	Jul 5, 2016

Abstract

A free-standing thin film is fabricated from a structure comprising a base layer coated with a sacrificial polymer layer, which is in turn coated with a flexible polymer layer. Cells are then seeded onto the flexible polymer layer and cultured to form a tissue. The flexible polymer layer is then released from the base layer to produce a free-standing thin film comprising the tissue on the flexible polymer layer. In one embodiment, the cells are myocytes, which can be actuated to propel or displace the free-standing film. In another embodiment, the free-standing film is used to treat injured human tissue.

First claim

What is claimed is:

1. A method for measuring contractility of a functional muscle tissue, comprising:
providing a free-standing muscle thin film, said free-standing muscle thin film comprising a flexible polymer layer, a spatially micro-patterned engineered surface chemistry deposited on the flexible polymer layer, and a functional muscle tissue;
attaching an end of the free-standing muscle thin film to a mounting structure;
applying a stimulus to cause the functional muscle tissue to contract; and
measuring the displacement of the muscle thin film when the functional muscle tissue contracts in response to the stimulus,
wherein the free-standing thin film is fabricated by
providing a base layer;
coating a sacrificial polymer layer on the base layer;
coating a flexible polymer layer that is more flexible than the base layer on the sacrificial polymer layer;
depositing a spatially micro-patterned engineered surface chemistry on the flexible polymer layer, wherein said spatially micro-patterned engineered surface chemistry allows for the alignment of muscle cells such that a functional muscle tissue is formed;
seeding muscle cells on the flexible polymer layer comprising the spatially micro-patterned engineered surface chemistry;
culturing the muscle cells to form a functional muscle tissue; and
releasing the flexible polymer layer from the base layer to produce the free-standing muscle thin film,

thereby measuring the contractility of the functional muscle tissue.

2. The method of claim 1 , wherein the base layer has an elastic modulus greater than 1 MPA.

3. The method of claim 1 , wherein the base layer is a glass cover slip.

4. The method of claim 1 , wherein the sacrificial polymer layer comprises poly (N-Isopropylacrylamide).

5. The method of claim 1 , wherein the flexible polymer layer comprises polydimethylsiloxane.

6. The method of claim 1 , wherein the muscle cells seeded on the flexible polymer layer are cardiomyocytes.

7. The method of claim 6 , wherein the cardiomyocytes are aligned to produce an anisotropic tissue.

8. The method of claim 1 , wherein the sacrificial polymer layer is coated on the base layer via spin coating.

9. The method of claim 1 , wherein the flexible polymer layer is coated on the sacrificial polymer layer via spin coating.

10. The method of claim 1 , wherein the sacrificial polymer is non-cross-linked poly(N-Isoproylacrylamide), and wherein the flexible polymer is released by dropping the temperature to 32° C. or less, causing the sacrificial polymer to liquefy.

11. The method of claim 1 , wherein the sacrificial polymer is crosslinked N-Isopopylacrylamide, and wherein the flexible polymer is released by dropping the temperature to 32° C. or less, causing the sacrificial polymer to become hydrophilic.

12. The method of claim 1 , wherein the sacrificial polymer is an electrically actuated polymer, and wherein the flexible polymer is released by applying an electric potential to the sacrificial polymer.

13. The method of claim 1 , wherein the sacrificial polymer is a degradable biopolymer, and wherein the flexible polymer is released by dissolving the sacrificial polymer.

14. The method of claim 1 , wherein the engineered surface chemistry comprises an extracellular matrix protein.

15. The method of claim 1 , wherein the engineered surface chemistry is provided in a pattern that includes gaps.

16. The method of claim 1 , wherein the cultured muscle tissue has a thickness of 200 micrometers or less when the flexible polymer layer is released.

17. The method of claim 1 , further comprising cutting the flexible polymer layer and the muscle tissue to produce a desired shape.

18. The method of claim 1 , wherein a radius of curvature of the muscle thin film is measured when the functional muscle tissue is contracted.

19. The method of claim 1 , wherein a rate of contraction of the functional muscle tissue is measured.

20. The method of claim 1 , further comprising contacting the muscle thin film with a candidate compound prior to applying the stimulus and comparing a degree of contraction or a rate of contraction of the functional muscle tissue in the presence of the candidate compound relative to displacement of the functional muscle tissue in the absence of the candidate compound, wherein a difference between the degree or rate indicates that the candidate compound alters muscle function.

21. The method of claim 1 , wherein the spatially micro-patterned engineered surface chemistry is a biopolymer.

22. The method of claim 1 , wherein the spatially micro-patterned engineered surface chemistry is selected from the group consisting of an extracellular matrix protein, a growth factor, a lipid, a fatty acid, a steroid, a sugar, a biologically active carbohydrate, a proteoglycan, a glycoprotein, a proteolipid, a glycolipid, a biologically derived homopolymer, a nucleic acid, a hormone, an enzyme, a pharmaceutical, a cell surface ligand, a cell surface receptor, a cytoskeletal filament, a cytoskeletal motor protein, and a hydrophylic polymer.

Assignees

• Harvard College

Inventors

• Parker Kevin Kit
• Feinberg Adam W
• Whitesides George M
• Shevkoplyas Sergey S
• Feigel Alexander

Classifications

• G01N2500/10

  involving cells · CPC title

• G01N33/5061 Primary

  Muscle cells · CPC title

• C12N5/0062

  General methods for three-dimensional culture · CPC title

• C12N2533/30

  Synthetic polymers · CPC title

• C12N5/0657

  Cardiomyocytes; Heart cells · CPC title