Compositions and methods for measuring cellular mechanical stress

Patent metadata

Field	Value
Publication number	US-10539552-B2
Application number	US-201515121733-A
Country	US
Kind code	B2
Filing date	Mar 4, 2015
Priority date	Mar 6, 2014
Publication date	Jan 21, 2020
Grant date	Jan 21, 2020

Abstract

Provided are compositions and methods employing cells encapsulated within and attached to a hydrogel, e.g., for measuring mechanical strain and/or stress of the cell and for investigating the mechano-chemo-transduction mechanisms at cellular and molecular levels.

First claim

What is claimed is:

1. A hydrogel composition comprising a cell encapsulated in a hydrogel, wherein the hydrogel is comprised of:
a first binding partner of a binding partner pair, wherein the first binding partner is avidin, and wherein the avidin is optionally conjugated to a biocompatible polymer,
a second binding partner of the binding partner pair, wherein the second binding partner is biotin, which is conjugated to a PEG polymer to form a biotin-PEG-biotin crosslinker,
wherein the avidin binds with the biotin-PEG-biotin crosslinker to form the hydrogel; and

wherein the cell is attached to a biotin-ligand conjugate via the ligand and the resultant biotin-ligand-cell conjugate is bound, via the biotin, to the avidin of the hydrogel, thereby encapsulating the cell in the hydrogel.

2. The composition of claim 1 , wherein the avidin is selected from avidin, streptavidin, neutravidin and captavidin.

3. The composition of claim 1 , wherein the biocompatible polymer is selected from the group consisting of polyvinyl alcohol (PVA), agarose and alginate.

4. The composition of claim 1 , wherein the hydrogel comprises a stiffness or Young's modulus value in the range of about 0.1 kPa to about 100 kPa.

5. The composition of claim 1 , wherein the hydrogel is optically transparent.

6. The composition of claim 1 , wherein the ligand binds to a cell surface molecule.

7. The composition of claim 6 , wherein the surface molecule is selected from the group consisting of an integrin, a glycan, a glycoprotein, and a cell surface receptor.

8. The composition of claim 1 , wherein the ligand is selected from a polypeptide, a linear peptide, a circularized peptide, a peptidomimetic, an antigen binding molecule, an antibody and an antibody fragment.

9. The composition of claim 1 , wherein the ligand is a polypeptide selected from the group consisting of collagen, laminin, fibronectin, fibrinogen, vitronectin, VCAM-1, cadherin, and subsequences thereof.

10. The composition of claim 1 , wherein the cell is a cardiac myocyte, a skeletal muscle myocyte or a smooth muscle myocyte.

11. A system comprising:
i) one or more perfusion chambers, each perfusion chamber comprising the hydrogel composition of any one of claims 1 , 2 , 3 , 4 , 5 , 6 - 9 , or 10 ;
ii) first and second mounting grips in contact with the hydrogel, wherein the first and second grips introduce tension on the hydrogel composition in at least two dimensions;
iii) a force transducer connected to the first grip and a tension manipulator connected to the second grip, wherein the force transducer senses mechanical tension imposed on the hydrogel composition which is used to calculate the stress in the cell and the hydrogel composition;
iv) a computer in communication with the force transducer, wherein the computer stores the resultant mechanical tension readings sensed by the force transducer.

12. A method of producing a composition of claim 1 , comprising:
a) incubating one or more cells with a biotin-ligand conjugate under conditions allowing the ligand to bind to one or more cell surface molecules on the one or more cells;
b) contacting the cells bound to the biotin-ligand conjugate with avidin and optionally conjugated to a biocompatible polymer under conditions sufficient for avidin to bind the biotin-ligand conjugate; and
c) contacting the cells from step b) with a biotin-PEG-biotin crosslinker under conditions sufficient to crosslink the avidin, thereby encapsulating the cells in a hydrogel.

13. A kit comprising:
i) a solution comprising a first binding partner of a binding partner pair, wherein the first binding partner is avidin, and wherein avidin is optionally conjugated to a biocompatible polymer;
ii) a solution comprising a Y-X-Y crosslinker; wherein X is a PEG polymer and Y is a second binding partner of the binding partner pair, wherein the second binding partner is biotin; and
iii) a solution of biotin conjugated to a ligand wherein the ligand is capable of binding to a cell.

Assignees

• Univ California

Inventors

• Chen-Izu Ye
• Lam Kit
• Izu Leighton T

Classifications

• C12M35/04

  Mechanical means, e.g. sonic waves, stretching forces, pressure or shear stimuli · CPC title

• G01N33/54366 Primary

  Apparatus specially adapted for solid-phase testing · CPC title

• C07K2319/24

  containing a MBP (maltose binding protein)-tag · CPC title

• C12M29/10

  Perfusion · CPC title

• C07K2319/21

  containing a His-tag · CPC title