Method and system for measurement of mechanical properties of molecules and cells

• US 5,486,457 A
• Filed: 08/25/1993
• Issued: 01/23/1996
• Est. Priority Date: 08/25/1993
• Status: Expired due to Term

• Current Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Original Assignee: Children's Medical Center Corporation, President and Fellows of Harvard College (Harvard Corporation)
• Inventors: Wang, Ning, Ingber, Donald E., Fredberg, Jeffrey J., Butler, James P.
• Primary Examiner(s): Scheiner, Toni R.
• Assistant Examiner(s): Wolski, Susan C.
• Application Number:
  US08/112,757
• Time in Patent Office: 881 Days
• Field of Search: 436/526, 436/806, 436/808, 435/7.2, 435/7.21, 435/7.23, 435/7.32, 435/149, 435/282, 435/291, 324/200, 324/228, 324/244
• US Class Current: 435/7.2
• CPC Class Codes: C12M 35/06 Magnetic means C12M35/02 takes precedence
• CPC Class Codes: C12M 41/36 of biomass, e.g. colony counters or by turbidity measurements electrooptical investigation of individual particles G01N15/14, flow cytometers G01N15/1404
• CPC Class Codes: C12M 41/46 of cellular or enzymatic activity or functionality, e.g. cell viability
• CPC Class Codes: G01N 24/00 Investigating or analyzing materials by the use of nuclear magnetic resonance, electron paramagnetic resonance or other spin effects
• CPC Class Codes: G01N 27/745 for detecting magnetic beads used in biochemical assays concerning the assays G01N33/54326; sensors therefor G01R33/1269; automatic analysers therefor G01N35/0098
• CPC Class Codes: G01N 35/0098 involving analyte bound to insoluble magnetic carrier, e.g. using magnetic separation magnetic particles used in immunoassays G01N33/54326; magnetic separation in general B03C
• CPC Class Codes: Y10S 436/806 Electrical property or magnetic property
• CPC Class Codes: Y10S 436/808 Automated or kit

Abstract

Mechanical stresses and deformations are applied directly to cell surface receptors or molecules and measured using a system including a magnetic twisting device in combination with ferromagnetic microbeads coated with ligands for integrins or any other surface receptors. The system can be used diagnostically to characterize cells and molecules and to determine the effect of transformation and compounds, including drugs, on the cells and molecules. The system can also be used to induce cells to grow or alter production of molecules by the cells.

Claims

1. A system for determining the effect of mechanical stimulation of molecules which are present on the surface of cells comprising:

ferromagnetic microbeads having attachment molecules coated thereon which bind to the molecules present on the surface of cells, the microbeads having a diameter between 0.03 and 500 microns, and wherein the attachment molecules are selected from the group consisting of matrix molecules that bind to integrins and molecules that bind to other receptors on the cell surface and physically interconnect with distinct cytoskeletal proteins, wherein the integrins or receptors are the molecules present on the surface of the cells to be tested;

an apparatus for imposing on the microbeads a strong external magnetic field resulting in magnetization and alignment of the magnetic moments of the microbeads and for imposing on the microbeads defined mechanical stresses between zero and 300 dyne/cm² using a weaker "twisting" magnetic field applied perpendicular to the original field; and

a magnetometer to measure the component of the remanent magnetic field vector in the direction of the original magnetization as a function of time.

2. The system of claim 1 wherein the strong external magnetic field is greater than 250 gauss and the weaker twisting magnetic field is between zero and 100 gauss.

3. The system of claim 1 wherein the attachment molecules are selected from the group consisting of fibronectin, vitronectin, collagens, laminin, fibrinogen, fibulin, dystrophin, heparan sulfate proteoglycan, plasminogen activator/urokinase, gangliosides, Von Willebrand'"'"'s factor, entactin, Echistatin, antibodies to integrins, synthetic RGD containing peptides, cadherins, and lectins.

4. The system of claim 1 wherein the cells are selected from the group consisting of plant cells, animals cells, yeast, and bacteria.

5. The system of claim 2 wherein the cells are living.

6. The system of claim 3 wherein the cells are permeabilized.

7. The system of claim 1 further comprising a compound to be tested for an effect on the molecules present on the surface of cells.

8. An in vitro method for detecting the effect of mechanical stimulation of molecules immobilized on a surface comprising:

mixing ferromagnetic microbeads having coated thereon attachment molecules which bind to the molecules immobilized on a surface, wherein the attachment molecules are selected from the group consisting of matrix molecules that bind to integrins and molecules that bind to other receptors that span the cell surface and physically interconnect with distinct cytoskeletal proteins, the microbeads having a diameter between 0.03 and 500 microns, with the molecules immobilized on a substrate;

treating the mixture of the microbeads and the immobilized molecules with a compound which is to be tested for an effect on the interaction of the attachment molecules on the microbeads with the immobilized molecules;

imposing on the microbeads a strong external magnetic field resulting in magnetization and alignment of the magnetic moments of the microbeads;

imposing on the microbeads defined mechanical stresses between zero and 300 dyne/cm² using a weaker "twisting" magnetic field applied perpendicular to the original field;

measuring the component of the remanent magnetic field vector in the direction of the original magnetization as a function of time;

comparing the effect of the compound to be tested on the interaction of the immobilized molecules with the attachment molecules on the microbeads with the effect of a compound having a predetermined effect on the interaction of the immobilized molecules with the attachment molecules on the microbeads.

9. The method of claim 8 wherein the strong external magnetic field is greater than 250 gauss and the weaker twisting magnetic field is between zero and 100 gauss.

10. The method of claim 8 wherein the attachment molecules are selected from the group consisting of fibronectin, vitronectin, collagens, laminin, fibrinogen, fibulin, dystrophin, heparan sulfate proteoglycan, plasminogen activator/urokinase, gangliosides, Von Willebrand'"'"'s factor, entactin, Echistatin, antibodies to integrins, synthetic RGD containing peptides, and cadherins.

11. The method of claim 8 wherein the substrate is cells selected from the group consisting of plant cells, animal cells, yeast, and bacteria.

12. The method of claim 11 wherein the cells are living.

13. The method of claim 11 wherein the cells are permeabilized.

14. The method of claim 8 wherein the molecules are immobilized on a non-cellular substrate.

15. The method of claim 11 further comprising repeating the method using cells of a second type and comparing the effect of the compound to be tested on the interaction of the attachment molecules on the microbeads with the molecules on the surface of the cells which were first tested with the interaction of the attachment molecules on the microbeads with the molecules on the surface of the second type of cells.

16. The method of claim 11 wherein cell functions are altered by the addition of compound.

17. The method of claim 16 wherein the cell function is growth or stiffness.

18. The method of claim 16 wherein the cell function is the production of molecules by the cells which is altered by exposure to the compound to be tested.

19. An in vitro method for detecting the effect of mechanical stimulation of molecules immobilized on cells comprising:

treating the cells with a compound which is to be tested for an effect on the interaction of the molecules immobilized on cells with attachment molecules coated on ferromagnetic microbeads, wherein the attachment molecules bind to the immobilized molecules and are selected from the group consisting of matrix molecules that bind to integrins and molecules that bind to other receptors that span the cell surface and physically interconnect with distinct cytoskeletal proteins, the microbeads having a diameter between 0.03 and 500 microns;

mixing the treated cells with the microbeads;

imposing on the microbeads a strong external magnetic field resulting in magnetization and alignment of the magnetic moments of the microbeads;

imposing on the microbeads defined mechanical stresses between zero and 300 dyne/cm² using a weaker "twisting" magnetic field applied perpendicular to the original field;

measuring the component of the remanent magnetic field vector in the direction of the original magnetization as a function of time;

comparing the effect of the compound to be tested on the interaction of the immobilized molecules with the attachment molecules on the microbeads with the effect of a compound having a predetermined effect on the interaction of the immobilized molecules with the attachment molecules on the microbeads.

20. An in vitro method for detecting the effect of mechanical stimulation of molecules immobilized on cells comprising:

mixing the cells with ferromagnetic microbeads having coated thereon attachment molecules which bind to the molecules immobilized on the cells, wherein the attachment molecules are selected from the group consisting of matrix molecules that bind to integrins and molecules that bind to other receptors that span the cell surface and physically interconnect with distinct cytoskeletal proteins, the microbeads having a diameter between 0.03 and 500 microns;

imposing on the microbeads a strong external magnetic field resulting in magnetization and alignment of the magnetic moments of the microbeads;

treating the mixture of the cells and the magnetized microbeads with a compound which is to be tested for an effect on the interaction of the molecules immobilized on cells with the attachment molecules on the microbeads;

imposing on the microbeads defined mechanical stresses between zero and 300 dyne/cm² using a weaker "twisting" magnetic field applied perpendicular to the original field;

measuring the component of the remanent magnetic field vector in the direction of the original magnetization as a function of time;

comparing the effect of the compound to be tested on the interaction of the immobilized molecules with the attachment molecules on the microbeads with the effect of a compound having a predetermined effect on the interaction of the immobilized molecules with the attachment molecules on the microbeads.

21. An in vitro method for detecting the effect of mechanical stimulation of molecules immobilized on cells comprising:

mixing the cells with ferromagnetic microbeads having coated thereon attachment molecules which bind to the molecules immobilized on the cells, and with a compound which is to be tested for an effect on the interaction of the molecules immobilized on the cells with the attachment molecules on the microbeads, wherein the attachment molecules are selected from the group consisting of matrix molecules that bind to integrins and molecules that bind to other receptors that span the cell surface and physically interconnect with distinct cytoskeletal proteins, the microbeads having a diameter between 0.03 and 500 microns;

imposing on the microbeads a strong external magnetic field resulting in magnetization and alignment of the magnetic moments of the microbeads;

imposing on the microbeads defined mechanical stresses between zero and 300 dyne/cm² using a weaker "twisting" magnetic field applied perpendicular to the original field;

measuring the component of the remanent magnetic field vector in the direction of the original magnetization as a function of time;

comparing the effect of the compound to be tested on the interaction of the immobilized molecules with the attachment molecules on the microbeads with the effect of a compound having a predetermined effect on the interaction of the immobilized molecules with the attachment molecules on the microbeads.