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USOO5486157A
United States Patent [191 [11] Patent Number: 5,486,457
Butler et al. (451 Date of Patent: Jan. 23, 1996
[54] METHOD AND SYSTEM FOR Bumdge, K., et al., “Focal Adhesions: Transmembrane
MEASUREMENT OF MECHANICAL Junctions Between The Extracellular Matrix and the Cytosk-
PROPERTIES OF MOLECULES AND CELLS eleton”, Ann. Rev. Cell Biol., 4:487-525 (1 988).
Cipriano, L. F., “An Overlooked Gravity Sensing
[75] Inventors: James P. Butler, Brookline; Jeffrey J. Mechanism”<The Physiologist, 34:72 (1991).
Fredberg, Sharon; Donald E. Ingber, De Groot, R. P., “Microgravity Decreases c-fos Induction
Boston; Ning Wang, Brookline, all of and Serum Response Element Activity”, J. Cell Sci., 97:33
Mass. (1990).
Dennerll, T. J., et al., “Tension and Compression in the
[73] Assignees: Children’s Medical Center Cytoskeleton of PC-12 Neurites 11: Quantitative Measure-
Corporation, Boston; President and ments”, J. Cell Biol. 107:665-674 (1988).
Fellows of Harvard College, Franke, R. P., et al., “Induction of Human Vascular Endot-
Cambridge, both of Mass. helial Stress Fibres by Fluid Shear Stress”, Nature,
307:648-649 (1984).
[21] Appl. No.: 112,757 Harris, R. C., et al., “Continuous Stretch-Relaxation in
Culture Alters Rat Mesangial Cell Morphology, Growth
[22] Filed: Aug. 25, 1993
Characteristcs, and Metabolic Activity”, Lab. Invest.,
[51] Int. C1.6 ..................................................... GOlN 33153 66(5):548554 (1992).
[52] US. C1. ......................... 435d7.2; 43517.21; 43517.23; Hay, M., et al., “Chromatic Motin in Neuronal Interphase
Nuclei: Changes Induced by Disruption of Intermediate
43517.32; 4351287.1; 4361526; 4361806;
Filaments”, Cell Motil. Cytoskel., 18:63 (1991).
4361808; 3241200; 3241228; 3241244
Ingber, D. E., “Fibronectin Controls Capillary Endothelial
[58] Field of Search ..................................... 4361526, 806,
Cell Growth by Modulating Cell Shape”, Proc. Natl. Acad.
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282, 291; 3241200, 228, 244
Ingber, D. E, et al., “How Does Extracellular Matrix Control
Capillary Morphogenesis?” Cell, 58:803-805 (1989).
[561 References Cited
(List continued on next page.)
PUBLICATIONS
Primary Examiner-Toni R. Scheiner
Ingber et al., “Integrins as mechanochemical transducers”,
Assistant ExaminerAusan C. Wolski
Current Opinion in Cell Biology, vol. 3, (1991), pp.
841-848. Attorney, Agent, or Fim-hall Golden & Gregory
Wang et al., “Mechanotransduction Across the Cell Surface [571 ABSTRACT
and Through the Cytoskeleton”, Science, vol. 260, (1993),
pp. 1124-1127. Mechanical stresses and deformations are applied directly to
Inber et al., ‘The Riddle of Morphogenesis: A Question of cell surface receptors or molecules and measured using a
Solution Chemistry or Molecular Cell Engineering?’, Cell, system including a magnetic twisting device in combination
VO~.7 5, (1993), pp.1249-1252. with ferromagnetic microbeads coated with ligands for
Wang et al., “Control of Cytoskeletal Mechanics by Extra- integrins or any other surface receptors. The system can be
cellular Matrix, Cell Shape, and Mechanical Tension”, Bio- used diagnostically to characterize cells and molecules and
physical Journal, vol. 66, (1994) pp. 1-9. to determine the effect of transformation and compounds,
Albelda, S. M., et al., “Integrins and Other Cell Adhesion including drugs, on the cells and molecules. The system can
Molecules”, FASEB J., 4:2868-2880 (1990). also be used to induce cells to grow or alter production of
molecules by the cells.
Bizal, C. L., et al., “Viscoelastic and Motile Properties of
Hamster Lung and Peritoneal Macrophages”, J. Leukocyte
Biol., 50:240 (1991). 21 Claims, 6 Drawing Sheets
16
0- 25 G
TWIST
7000 G
(1 MINUTE1 18
PULSE
10 ,12 MAGNETOMETER
BEFORE AFTER DURING
MAGNETIZATION MAGNET 12 AT ION TWIST
5,486,457
Page 2
PUBLICATIONS Biol., 26:905 (1990).
Schwartz, M. A., et al., “Insoluble Fibronectin Activates the
Ingber, D. E., et al., “Mechanochemical Switching Between
Na/H Antiporter by Clustering and Immobilizing Integrin
Growth and Differentiation During Fibroblast Growth
a,@,,I ndependent of Cell Shape”, Proc. Natl. Acad. Sci,
Factor-Stimulated Angiogenesis In Vitro: Role of Extracel-
U.S.A., 88:7849-7853 (1991).
lular Matrix”, J. Cell Biol., 109:317-330 (1989).
Sims, J. R., et al., “Altering the Cellular Mechanical Force
Janmey, P. A., et al., “Viscoelastic Properties of Vimentin
Balance Results in Integrated Changes in Cell, Cytoskeletal
Compared with Other Filamentous Biopolymer Networks”,
and Nuclear Shape”, J. Cell Sci., 103:1215-1222 (1992).
J. Cell Biol., 113:155-160 (1991).
Sumpio, B. E., et al., “Enhanced Collagen Production by
Kacahr, B., et al., “Structural Basis for Mechanical Trans-
Smooth Muscel Cells During Repetitive Mechanical
duction in the Frog Vestibular Sensory Apparatus: I. The
Stretching”, Arch. Surg. 123:1233 (1988).
Otolithic Membrane”, Hearing Res., 45: 179 (1990).
Terracio, L., et al., “Effects of Cyclic mechanical Stimula-
Kolega, J., “Effects of Mechanical Tension on Protrusive
tion of the Cellular Components of the Heart: In Vitro”, In
Activity and Microfilament and Intermediate Filament
Vitro Cell Dev. Biol., 2453 (1988).
Organization in an Epidermal Epithelium Moving in Cul-
Valberg, P. A,, et al., “Magnetic Particle Motions Within
ture”, J. Cell Biol., 102:1400-1411 (1986).
Living Cells- Physical Theory and Techniques”, Biophys.
Komuro, I., et al., “Mechanical Loading Stimulates Cell
J., 52~537-550 (1987).
Hypertrophy and Specific Gene Expression in Cultured Rat
Cardiac Myocytes”, J. Biol. Chem., 266:1265-1268 (1991). Valberg, P. A., “Magnetometry of Ingested Particles in
Lansman, J. B., “Single Stretch-activated Ion Channels in Pulmonary Macrophages”, Science, 2245 13-516 (1984).
Vascular Endothelial Cells as Mechanotransducers?”, Valberg, P. A., et al., “Cytoplasmic Motions, Rheology, and
Nature, 325:811-813 (1987). Structure Probed by a Novel Magnetic Particle Method”, J.
Moller, W., et al., “Improved Spinning Top Cell Biol, 101:13&140 (1985).
Aerosol-generator for the Production of High Concentrated Wagner, V. T., et al., “Role of a Vitronectin-like Molecule in
Ferrimagnetic Aerosols”, J. Aerosol Sci., 21:S657 (1990). Embryo Adhesion of the Brown Alga Fucus”, Proc. Natl.
Murti, K. G., et al., “Protein Kinase C Associates with Acad. Sci. U.S.A., 89:3644-3648 (1992).
Intermediate Filaments and Stress Fibers”, Exp. Cell Res., Watson, P. A., “Direct Stimulation of Adenylate Cyclase by
202:364 (1992). Mechanical Forces in S49 Mouse Lymphoma Cells During
Olesen, S.- P., et al., “Haemodynamic Shear Stress Activates Hyposmotic Swelling”, J. Biol. Chem., 265:6569-6575
a K” Current in Vascular Endothelial Cells”, Nature, (1990).
331: 168-170 (1988). Wayne, R., et al., “The Contribution of the Extracellular
Ryan, T. J., “Biochemical Consequences of Mechanical Matrix to Gravisensing in Characean Cells”, J. Cell Sci.,
Forces Generated by Distention and Distortion”, J. Am. 101:611 (1992).
Acad. Derm., 21:115 (1989). Wilson, L. J., et al., “Functional Morphology of the
Sachs, F., “Ion Channels as Mechanical Transducers”, Cell Telson-Uropod Stretch Receptor in the Sand Crab Emerita
Shape: Determinants, Regulation, and Regulatoly Role, Analoga”, J. Comp. Neurol., 296:343-358 (1990).
63-92 (9189). Wirtz, H. R. W., et al., “Calcium Mobilization and Exocy-
Samuel, J.-L., et al., “Mechanically Induced Orientation of tosis After One Mechanical Stretch of Lung Epithelial
Adult Rat Cardiac Myocytes In Vitro”, In Vitro Cell Dev. Cells”, Science, 250:1266-1269 (1990).
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 1 of 6
U
0
c
h
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 2 of 6
-
90
m
BSA- bead
f
AcLDL - bead
-Q)
U
60 D RGD- bead +
GRGDSP
oRGD- bead + Cyt
Ab-01- bead
RGD- bead
ZI:
Q
Stress (dynes/cm2)
FIG 2
-
100
- F R G D -
bead
80- Acr
-
NOC
60-
-
40-
-
CyT Acr
+
20- Cy1 Noc
+
-
1 I 1 I I 1 1 I I
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 3 of 6
60
50-
h
Y
0 10 20 30 40 50
Stress (dyne/cm2)
FIG 4a
0 1 I 1 1 I
0 10 20 30 40 50
Stress (dyne /cm 2 1
FIG. 46
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 4 of 6
80
60
40
20
0
LOW FN HIGH FN
FIG 5
150 1 I
T
100
50
0
n.c-
E
cv
Y
IL
ki
+
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 5 of 6
120
7-
100
80
FIG 7
60 I
40
T
20
0
a
La w
0
sm 0 0
c
m
U
0
f 73
t S
0 v) 3
0 W 2
E 2
m m
150
130
FIG 8
110
90
0.0 0.5 1.5
AGM (TNP-47d*?tng/rnl)
U.S. Patent 5,486,457
Jan. 23,1996 Sheet 6 of 6
100-
-
80
-
60
40 -
-
20
L
0 -
0
L
t
Iz
0
0
200
BREAST CANCE
150
100 FIG. 9b
50
0
-
0
L
t
c
8
5,486,457
1 2
METHOD AND SYSTEM FOR culture substrata alters CSK organization and induces bio-
MEASUREMENT OF MECHANICAL chemical changes in adherent cells, reported by Wirtz and
PROPERTIES OF MOLECULES AND CELLS Dobbs, Science 250,1266 (1990); Samuel and Vandenburgh,
In Vitro Cell Dev. Biol. 26, 905 (1990); Harris, et al., Lab.
BACKGROUND OF THE INVENTION 5 Invest. 66,548 (1992); Sumpio, et al., Arch. Surg. 123, 1233
(1988); Terracio, et al., In Vitro Cell Dev. Biol. 24,53 (1988).
The United States government has rights in this invention
However, in these and other stretching studies, it is not
by virtue of NASA grant No. NAG-9-430 and NIH grant
possible to separate effects due to transmembrane force
Nos. CA4554B to Donald Ingber, NIH grant No. HL33009
transfer from those due to global shape changes and gener-
to Jeffrey J. Fredberg and NIH grant No. HL36427 to James
10 alized deformation of the plasma membrane and CSK.
P. Butler.
It is therefore an object of the present invention to provide
The process of recognizing and responding to mechanical
a method and system for applying controlled mechanical
stimuli is critical for growth and function of living cells.
loads directly to specific molecules, either isolated or
Many sensory functions including touch, hearing, barore-
expressed on cell surfaces, for characterizing molecules and
ception, proprioception, and gravity sensation involve spe-
cialized mechanotransduction mechanisms. Development of l5 cells and their properties.
tissue pattern is also exquisitely sensitive to changes in It is a further object of the present invention to provide a
mechanical stress. Nevertheless, the molecular mechanism method and system for applying controlled mechanical loads
by which individual cells recognize and respond to external directly to specific molecules to test compounds potentially
forces is not well understood. Stretch-sensitive ion channels, 2o affecting molecules and cells to determine if the compounds
adenylate cyclase, and protein kinase C change their activity affect the mechanical properties of the molecules and cells,
in response to applied stress, as reported by E Sachs, in Cell and the extent of this affect.
Shape: Determinants, Regulations, and Regulatory Role, W. It is another object of the present invention to provide a
D. Stein and E Bronner, eds. (Academic Press, San Diego, method and system which can separate effects due to specific
1989), pp. 63-92; 1. Komuro et al., J. Biol. Chem. 266,1265 25 transmembrane force transfer from those due to global shape
(1991); R. P. De Groot et al., J. Cell Sci. 97, 33 (1990); S. changes and generalized deformation of the plasma mem-
P. Olesen, et al., Nature 331, 168 (1988); J. B. Lansman, brane and cytoskeleton.
Nature 325,811 (1987); P. Watson, J. Biol. Chem. 265,6569 It is another object of the present invention to provide a
(1990); andT. J. Ryan, J. Am. Acad. Derm. 21, 115 (1989). method and system which can assess the status of the cell
However, these signaling pathways are likely to lie down- 30 with respect to the mechanical properties, strength, shape,
stream from the initial mechanoreception event at the cell stiffness, rheology, proliferation, and other factors relevant
surface. For example, activation of these signaling mol- to the health and function of the cell.
ecules appears to be mediated through changes in the
cytoskeleton (CSK), as reported by Komuro et al.; T. J.
Ryan, J. Am. Acad. Derm. 21,115 (1989); K. G. Murti, et al.,
35 SUMMARY OF THE INVENTION
Exp. Cell Res. 202, 36 (1992). While changes in CSK
organization are an ubiquitous response to mechanical per- Mechanical stresses are applied directly to specific mol-
turbation, B. Kacahr, et al., Hearing Res. 45, 179 (1990); L. ecules, either within or as expressed on cell surfaces or on
J. Wilson and D. H. Paul, J. Comp. Neurol. 296,343 (1990); non-cellular substrates, using a system including a magnetic
L. E Cipriano, The PhysioZogist 34, 72 (1991); T. J. Den- 40 twisting device in combination with ferromagnetic micro-
nerll, et al., J. Cell Biol. 107, 665 (1988); J. Kolega, J. Cell beads coated with attachment molecules. Examples of mol-
Biol. 102, 1400 (1986); R. P. Franke et al., Nature 307,648 ecule-specific attachment molecules include ligands for inte-
(1984), the mechanism by which forces are transmitted grins (e.g., extracellular matrix molecules, synthetic ECM
across the cell surface and transduced into a CSK response peptides, and anti-integrin antibodies), and other molecules
remains unknown. 45 binding to non-integrin surface-bound molecular receptors
Previous analysis of mechanotransduction used standard (for example, ligands for cell-cell adhesion receptors “cad-
methods to apply mechanical strain (stretch) or compressive herins”, that also link up to the cytoskeleton). “Non-spe-
loads and associated generalized deformation to whole cells cific” ligands can also be used as attachment molecules.
and tissues in specialized force-sensing cells. These studies, Cells can be living or dead, intact or permeabilized. The
in both plants and animals, suggest that the cell’s extracel- 50 cells or isolated molecules are immobilized so that their
lular matrix (ECM) attachments are the sites at which forces interaction with the ferromagnetic beads can be manipulated
are transmitted to cells, see, for example, Kacahr, et al., by application of magnetic forces.
(1990); Wilson and Paul, (1990); Wagner, et al., Proc. Natl. The system is used (1) to apply stresses to cells without
Acad. Sci. U.S.A. 89, 3644 (1992); R. Wayne, et at., J. Cell inducing global shape change, (2) to measure those stresses,
Sci. 101, 611 (1992); D. E. Ingber, Cur,: Opin. Cell Biol. 3, 55 (3) to measure resulting (local) distortions, and (4) to
841 (1991). As in any architectural structure, mechanical measure changes in these quantities using a wide variety of
loads are transmitted across the cell surface and into the cell biological interventions, protocols and circumstances. In the
by means of structural elements that are physically inter- simplest case, one can measure the avidity of protein-protein
connected. Transmembrane ECM receptors, such as mem- binding by quantitating the ability of the bound complex to
bers of the integrin family, are excellent candidates for 60 resist m~~ echanical perturbation (twisting). This approach can
mechanoreceptors because they bind actin-associated pro- be used to screen for high aflinity ligands or to quantitate the
teins within focal adhesions and thereby physically link mechanical properties (stiffness or elasticity, permanent
ECM with CSK microfilaments, as reviewed by S. M. deformation, viscosity or rheology) of synthetic or naturally
Albelda and C. A. Buck, FASEB J. 4, 2868 (1990); K. produced materials, fabrics, filters, etc. In a more complex
Burridge, et al., Ann. Rev. Cell Biol. 4, 487 (1988). The 65 case, one can measure the mechanical properties of intact
possibility that ECM receptors mediate mechanotransduc- living cells, by twisting specific molecules that are exposed
tion is supported by the finding that stretching flexible ECM on the cell surface and are physically interconnected with
5,486,457
3 4
the molecular scaffoldings, or cytoskeleton, that form the DETAILED DESCRIPTION OF THE
structural backbone of the cell. INVENTION
The system can be used diagnostically to characterize
cells and to determine the effect of transformation and
The System
compounds, including drugs, on the cells, thereby forming 5
the basis of a screen for useful modulators of cell shape, To determine whether any particular receptor system,
growth and function. The system can also be used to induce such as ECM receptors, provide a specific molecular path for
gene expression, alter production of molecules by the cells, mechanical signal transfer to the cytoskeleton (CSK), a
or mechanically disrupt membrane continuity and thereby method was devised in which controlled mechanical loads
permit transmembrane delivery of large molecules. 10 can be applied directly to specific cell surface molecules
without producing global changes of cell shape. The method
is shown schematically in FIG. 1. In this device, these loads
BRIEF DESCRIPTION OF THE DRAWINGS
can not only be applied, but also the load and the resulting
FIG. 1 is a schematic of the method using the magnetic deformation measured.
twisting device. l5 As shown by FIG. 1, in one embodiment, ferromagnetic
FIG. 2 is a graph of the stress-strain relation measured microbeads 10 coated with molecular ligands are allowed to
using magnetic microbeads attached to the surfaces of living bind to their CorresPonding molecular receptors on the
cells. Applied stress was determined by a calibration tech- surfaces Of cells 12 for 10 to 15 minutes and unbound beads
nique in which the Same beads were twisted in a standard 2o are removed before magnetic manipulation is initiated. Cells
solution of known viscosity of 1000 poise. Angular strain 12 are adhered to plastic dishes 11, or otherwise immobi-
(bead rotation) was calculated as the arc cosine of the ratio lized, for example, within a gel matrix. In a second embodi-
of remanent field after 1 d nt wist to the field at time 0. ment (not shown), molecules are adhered to or immobilized
Angular strain is plotted here as degrees. Bead coating: on a surface or within a volume other than a cell and bound
RGD, Arg-Gly-Asp-containing synthetic peptide; Ab.$,; 25 by the attachment molecules on the ferromagnetic beads.
AcLDL, acetylated-low density lipoprotein; BSA, bovine Brief application of a strong exkrnal magnetic field 14
serum albumin. GRGDSP, soluble fibronectin peptide (1 (1000 Gauss for 10 P) results in magnetization and align-
mg/& added for 10 min); Cyt, cytochalisin D (0.1 pg/d). ment of the magnetic moments of all surface-bound beads
BSA-bead: inverted triangle; AcLDL-bead, closed square; 10. Defined mechanical stresses (0-100 dynes/cm2, prefer-
RGD-bead+GRGDSP, open triangle; RGD-beadcyt, open 3o ably 0-68 dYnes/cm2) are then applied without remagnetiz-
circle; Ab-pl-bead, open square; RGD-bead, closed circle. ing the beads using a Weaker “twisting” magnetic field 16
Error bars, SEM. (0-100 Gauss, preferably 0-25 Gauss) applied perpendicu-
to the original field. The average bead rotation (angular
FIG. 3 is a graph of the stiffness (dynes/cm2) of the CSK
strain) induced by the twisting field is quantitated using a
of living cells, defined as the ratio of stress to strain (in
radians) at 1 fin twisting. Not, nocodazole (10 pg/d); Acr, 35 magnetometer l8t o changes in the component Of
acrylamide (4 m);C yt, cytochalasin (0.1 pg/dR)G. D- the remanent magnetic field vector in the direction of the
original magnetization as a function of time. In the absence
bead, closed circle; open triangle; Not, open square;
of force transmission across the cell surface, the spherical
Cyt, open circle; Cyt+Acr, inverted triangle; c ~ ~da+rk ~ ~ ~ ,
beads would twist in place by 90” into complete alignment
square.
with the twisting field, and the remanent field vector would
FIGS. 4a and 4b are graphs of the
strain and 4~ immediately drop to zero. In contrast, transmission of force
Of round On low fibronectin, lon glcm2> to the CSK would result in increased resistance to deforma-
(open circles) and spread cells (cells on high fibronectin, 500
tion and decreased bead rotation,
ng/cm2) (closed circles) as stress was applied from 7 to 40
dyne/cm2: FIG. 4a, angular strain (degree) versus stress This system was described
(dyne/cm2); FIG. 4b, stiffness (dyne/cm2) versus stress 45 by et al., J. LeukoBqiotz.o 5 0, 240 (lggl);
and Butler, Biophys. J. 52, 537 (1987); Valberg and Alber-
(dyne/cm2).
tini, J. Cell Biol. 101, 130 (1985); and Valberg, Science
FIG. is a bar graph Of the deformation (%) 2224, 513 (1984), for use in measuring viscoelasticity in
for round On low fibronectin7 lo ndcm2) and living cells that ingested or injected ferromagnetic
spread cells (cells on high fibronectin, 500 ng/cm2), after the 5o particles.
applied stress (40 dyne/cm2) was removed.
As described herein, this system is modified such that
FIG’ is a Paph Of the (poise) Of ferromagnetic microbeads are coated with specific receptor
spread Cells (high fibronectin), round cells (low fibronectin),
ligands, for example, which mediate or spread-
saponin membrane permeabilized spread cells without ATP
ing, which are bound to molecules on the external surface of
(after Perm)’ minutes after adding ATP to permeabilized 55 or specific molecules within the cells to be characterized. By
min)y and 2o IninUtes after adding ATP to magnetizing these surface-bomd beads in one direction and
permeabilized cells (+ATP, 20 min).
then applying a second weaker magnetic field oriented at
FIG. 7 is a graph ofthe stiffness (dYnes/cm2) for spread go”, it is possible to twist the beads in place and thereby
bovine capillary (BCE) cells, bovine Pulmonary exert a controlled shear stress (0-1 00 dynes/cm2, preferably
arterial smooth muscle cells, breast cancer cells, and round 6o 0-68 dynes/cm2) on bound cell surface receptors. An in-line
BCE. magnetometer is used to simultaneously measure changes in
FIG. 8 is a graph showing the stiffness (% of control) for the orientation of the magnetized beads and hence, quanti-
control BCE and BCE treated with TNP-470. tate angular strain produced in response to the applied stress.
FIGS. 9a and 9b are graphs of the stiffness (% of control) This system can be altered by varying the amplitude of the
for control BCE and BCE treated with 15 pM taxol (FIG. 9a) 65 twisting magnetic fields using any desired time history, such
and control breast cancer cells and breast cancer cells treated as a square wave or sine wave function. The device can also
with 15 pM taxol (FIG. 9b). be modified to allow systematic probing with magnetic
