1 Cell Biomechanics and Methods Z. Huang, A. Pettes, and B. Pruitt August 2, 2007

2 Motivation Balaban et al, 2001 Force generation vectors Cardiac myocyte Understanding mechanical effects on cellular processes and events triggered by mechanical stimuli (mechanotransduction) Determining the exact nature of mechanotransduction in –cardiac myocytes –sense of touch –bone remodeling Stress environment –an important parameter in tissue engineering

3 Motivation

4 Applied External Force Mechano-sensitive Ion Channel signaling cascade change in protein synthesis or post- translational modifications conformal changes in structural proteins altered gene expression

5 Motivation Forces are transmitted through adhesion sites attached to the cytoskeleton Deformation causes the cells to react – Apoptosis, cell-division, growth Applications –Sense of Touch –Cancer growth –Hypertension –Bone remodeling –Heart attack due to blood clots –Regenerative medicine

6 Biology Force Measurements TechniqueForce RangeAuthorsYear Antibody-Antigen Binding100 pNLee and Colton1994 Cellular Adhesion5-100 pNAdams, Chen, Smith and Nelson 1998 Saif2003 Actin-Myosin Mechanical Behavior 10 pN-10nNFiner, Simmons and Spudich 1994 Protein Folding pNPowers, Daburesse, Noller 1993 Further research is necessary to develop more precise models of cellular behavior, drug development, testing, and manufacturing.

7 Cell Patterning AFM of NIH 3T3 Vinculin Plaques K. Parker focal adhesions

8 Cell Patterning Devices to apply controlled forces / strains to live cells and study the response Estimate the cell stiffness and monitor changes due to loading effects Correlate response with cytoskeleton dynamics and signaling molecules Dynamically measure effects of cyclic loading and varying strain rates

9 Cell Patterning Pattern a binding agent (e.g. Fibronectin) through soft lithography to confine cell movement and study isolated and/or patterned arrays of cells Whitesides et al, 2001

10 Available Measurement Techniques Van Vliet, 2003 Cell Populations SD - Substrate Deformation Cell Populations / Single Cells EPT - Embedded Particle Tracking MTC - Magnetic Twisting Cytometry mPAD - microfabricated Post Array Detector Single Cells CD – Cytodetacher MA - Micropipette Aspiration OS - Optical Stretcher Single Cells / Molecules AFM - Atomic Force Microscopy BFP - Biomembrane Force Probe HRFS – High Resolution Force Spectroscopy MN - Microneedles MT - Magnetic Tweezer OT - Optical Trap Force [N] Distance [m]

11 Forces in Single Cells Cell Populations SD - Substrate Deformation Cell Populations / Single Cells EPT - Embedded Particle Tracking MTC - Magnetic Twisting Cytometry mPAD - Microfabricated Post Array Detector Single Cells CD – Cytodetacher MA - Micropipette Aspiration OS - Optical Stretcher Single Cells / Molecules AFM - Atomic Force Microscopy BFP - Biomembrane Force Probe HRFS – High Resolution Force Spectroscopy MN - Microneedles MT - Magnetic Tweezer OT - Optical Trap Van Vliet, 2003 Force [N] Distance [m]

12 Micropatterned Substrates mPAD Tan et al., 2003 Beningo et al., 2003 EPT

13 Micropost Array Studies mPAD Chen et al., 1997 mPAD

14 Magnetic Twisting Cytometry (MTC) Morales et al., 2001

15 Properties of Cellular and Sub-cellular Components Cell Populations SD - Substrate Deformation Cell Populations / Single Cells EPT - Embedded Particle Tracking MTC - Magnetic Twisting Cytometry mPAD - microfabricated Post Array Detector Single Cells CD – Cytodetacher MA - Micropipette Aspiration OS - Optical Stretcher Single Cells / Molecules AFM - Atomic Force Microscopy BFP - Biomembrane Force Probe HRFS – High Resolution Force Spectroscopy MN - Microneedles MT - Magnetic Tweezer OT - Optical Trap Van Vliet et al., 2003 Force [N] Distance [m]

16 Micropipette Aspiration (MA) Jones et al., 1999

17 Atomic Force Microscopy (AFM) Van Vliet et al., 2003 AFM

18 Optical Trapping (OT) Dao et al., 2003

19 Stimuli and Response in Cell Populations Cell Populations SD - Substrate Deformation Cell Populations / Single Cells EPT - Embedded Particle Tracking MTC - Magnetic Twisting Cytometry mPAD - microfabricated Post Array Detector Single Cells CD – Cytodetacher MA - Micropipette Aspiration OS - Optical Stretcher Single Cells / Molecules AFM - Atomic Force Microscopy BFP - Biomembrane Force Probe HRFS – High Resolution Force Spectroscopy MN - Microneedles MT - Magnetic Tweezer OT - Optical Trap Other Techniques Deformable Substrates Flow Shear Stress Van Vliet et al., 2003 Force [N] Distance [m]

20 Stimuli and Response in Cell Populations Deformable Substrates Feeback and Clarke, 2000 Usami et al., 1993 Flow Shear Stress

21 MEMS Cell-Force Measurments Nelson et al, 2005

22 MEMS Cell-Force Measurments Nelson et al, 2005

23 MEMS Heart Cell-Force Transducer Lin et al., 2000

24 Cell-Force Measurements in Malaria-affected Cells Healthy red blood cell ↓Exposed to P. ↓falciparum parasite Uninfected Ring stage Trophozoite stage Schizont stage Suresh et al., 2005

25 Thank you