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Nikolai Eroshenko BBSI: Summer 2006 Dr. Taylor’s Nanomanufacturing Laboratory

Using mechanical cues for controlled stem cell differentiation: Quantification of material properties of PDMS. Nikolai Eroshenko BBSI: Summer 2006 Dr. Taylor’s Nanomanufacturing Laboratory Dr. Rao’s Stem Cell Laboratory. Embryonic development. Cells in a developing embryo.

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Nikolai Eroshenko BBSI: Summer 2006 Dr. Taylor’s Nanomanufacturing Laboratory

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  1. Using mechanical cues for controlled stem cell differentiation:Quantification of material properties of PDMS Nikolai Eroshenko BBSI: Summer 2006 Dr. Taylor’s Nanomanufacturing Laboratory Dr. Rao’s Stem Cell Laboratory

  2. Embryonic development

  3. Cells in a developing embryo mechanical properties of extracellular matrix (ECM) genes hormones cell behavior chemical signals intercellular interactions

  4. Normal branching morphogenesis in embryonic mouse lung

  5. Normal branching morphogenesis in embryonic mouse lung epithelial cell monolayer basement membrane (ECM)

  6. Normal branching morphogenesis in embryonic mouse lung epithelial cell monolayer basement membrane (ECM) local increase in ECM turnover

  7. Normal branching morphogenesis in embryonic mouse lung epithelial cell monolayer basement membrane (ECM) Cell division is paralleled with basement membrane deposition

  8. Normal branching morphogenesis in embryonic mouse lung epithelial cell monolayer basement membrane (ECM)

  9. Normal branching morphogenesis in embryonic mouse lung epithelial cell monolayer basement membrane (ECM)

  10. Elasticity of ECM determines shape of endothelial, epithelial, and fibroblast cells rigid substrate soft substrate

  11. Shape of cells determines their fate DEATH

  12. Research question • Just how stiff or soft do the substrates have to be for the cells to assume the various morphologies? • How sensitive are cells to local changes in substrate stiffness?

  13. Primary goals • Quantitate the substrate elasticity values that induce morphological changes in different human cell types. • Determine how sensitive different cell types are to gradients of substrate rigidity. • Use mechanical cues to guide human embryonic stem cell lineage commitment.

  14. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create controlled stiffness gradients

  15. Choosing a material • Previous studies have used: • polyacrylamide • polydimethylsiloxane (PDMS)

  16. Acrylamide vs PDMS Polyacrylamide PDMS

  17. Acrylamide vs PDMS Polyacrylamide PDMS

  18. Choosing a material • Previous studies have used: • Polyacrylamide • greater crosslinker diffusion • genotoxic • sends chemical cues to cells • polydimethylsiloxane (PDMS) • does not appear to have significant crosslinker diffusion • no published evidence of genotoxicity • slightly ionic surface

  19. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create controlled stiffness gradients

  20. Ways to measure material properties • Atomic force microscope (AFM): • imaging • topography • Nanoindenter • elasticity

  21. Atomic force microscope • Sharp tip – radius on the order of 10nm • Can apply a wide range of forces: nN-10,000μN • Hard to know the tip shape • Hard to accurately determine the probe spring constant

  22. Nanoindenter transducer • Can accurately measure nm-scale indent depths • Load resolution of ~±100nN • Tip radius 80-120nm • Cannot detect initial contact forces of <1μN • Not capable of taping mode; only scanning probe microscopy diamond tip

  23. insulating housing nanoindenter video optics piezo scanner transducer nanoindenter tip XY stage

  24. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create controlled stiffness gradients

  25. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create controlled stiffness gradients

  26. Finding the elasticity of PDMS • Measuring of elastic modulus of PDMS created with different crosslinker-to-base ratios • Measuring of elastic modulus PDMS cured at different temperatures depth, load tip area function area of tip-material contact Elasticity Oliver-Pharr method E=(F/A0)/(Δl/l0)

  27. Preliminary nanoindentation data • Tests of 10-1 base-to-crosslinker mixture: • E=2.6±.4 MPa • Result agrees with recent study: • E=2.04 MPa (unconfined compression) • E=3.64 MPa (Oliver and Pharr model) • E=.58 MPa (Derjaguin-Muller-Toporov model) Carrillo F, Gupta S, Balloch M, Marshall SJ, Marshall GW, Pruit L, Puttlitz CM: Nanoindentation of polydimethylsiloxane elastomers: Effect of crosslinking, work of adhesion, and fluid environment on elastic modulus. Journal of Material Research 20.10: 2820-2830 (2005).

  28. Preliminary nanoindentation data • Creep due to viscoelasticity • Increase in noise at rates >~1000nm/s ANOVA: r2=0.434

  29. Preliminary Nanoindentation Data • Confirms the presence of creep • Total indentation time matters ANOVA (200nm/s): ANOVA (300nm/s): r2(200nm/s)=0.922 r2(300nm/s)=0.942

  30. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create a controlled stiffness gradient

  31. Proposed microfluidics design

  32. Cells Biomaterials • Learn the basics of human tissue culture • Learn to work with • fibroblasts • neuron cells • endothelial cells • embryonic stem cells • Study adult human cells on substrates of various rigidities • Study adult human cells on substrates with various rigidity gradients • Study stem cell differentiation on substrates with various rigidities • Choose a material • Find ways to measure mechanical properties • Learn to use the testing instruments • Find the optimal testing protocols • Test material • Design a microfluidics system to test diffusion and create controlled stiffness gradients

  33. QUESTIONS? For the curious: • Ingber DE: Mechanical control of tissue morphogenesis during embryological development. Int. J. Dev. Biol. 50:255-266(2006). • Carrillo F et al.: Nanoindentation of polydimethylsiloxane elastomers: Effects of crosslinking, work adhesion, and fluid environment on elastic modulus. J. Mater. Res. 20.10:2820-2830(2005) • Yeung T et al.: Effects of substrate stiffness on cell morphology, cytoskeletal structure, and adhesion. Cell Motil. and the Cyt. 60:24-34(2005). • McBeath R et al.: Cell shape, cytoskeletal tension, and RhoA regulate stem cell lineage commitment. Devel. Cell 6:483-495(2004) • Lo C-M et al.: Cell movement is guided by rigidity of the substrate. Biophys. J. 79:144-152(2000). • Huang S, Ingber DE: Shape-dependent control of cell growth, differentiation, and apoptosis: Switching between attractors in cell regulatory networks. Exper. Cell Res. 261:91-103(2000). • Oliver WC, Pharr GM: An improved technique for determining hardness and elastic modulus using load and displacement sensing indentation experiments. J. Mater. Res. 7.6:1564-1583(1992).

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