Micro Patterning for Biological Applications

Micro Patterning for Biological Applications PowerPoint PPT Presentation


  • 161 Views
  • Uploaded on
  • Presentation posted in: General

Download Presentation

Micro Patterning for Biological Applications

An Image/Link below is provided (as is) to download presentation

Download Policy: Content on the Website is provided to you AS IS for your information and personal use and may not be sold / licensed / shared on other websites without getting consent from its author.While downloading, if for some reason you are not able to download a presentation, the publisher may have deleted the file from their server.


- - - - - - - - - - - - - - - - - - - - - - - - - - E N D - - - - - - - - - - - - - - - - - - - - - - - - - -

Presentation Transcript


1. Micro Patterning for Biological Applications IGERT/HHMI Journal Club April 23, 2007 Matt Keuss ZiQiu Tong

2. History patterning of species on surfaces with micron scale resolution 1978, MacAlear from semiconductor industry Integration of protein molecules into bioelectronic microcircuits

3. Potential Applications Celluar studies Tissue engineering Development of biosensors Immunoassay Cellular patterns are used to address fundamental issues in cell biology, like cell-cell, cell-substrate and cell-medium interactions. Tissue engineering? patterning of 2 or more cell types in a co-culture sysytem allows mnanupulation of cell-cell interaction which has potential in tissue engineering. The accurate positioning of cells and biomolecules is essential for the development of cell and molecular-based biosensors. Cellular patterns are used to address fundamental issues in cell biology, like cell-cell, cell-substrate and cell-medium interactions. Tissue engineering? patterning of 2 or more cell types in a co-culture sysytem allows mnanupulation of cell-cell interaction which has potential in tissue engineering. The accurate positioning of cells and biomolecules is essential for the development of cell and molecular-based biosensors.

4. Micropatterning Requirements Selective attachment of protein at desired regions High protein resistivity by other regions Protein orientation – immunodiagnostic devices Retaining protein functionality Main requirement for protein patterning is the selective attachment of protein at the desired regions and high protein resistivity by other regions. Orientation is important in the development of immuodiagnostic devices Retaining protein functionality is crucial for ensuring that protein will serve its purpose and provide reliable analysis. Main requirement for protein patterning is the selective attachment of protein at the desired regions and high protein resistivity by other regions. Orientation is important in the development of immuodiagnostic devices Retaining protein functionality is crucial for ensuring that protein will serve its purpose and provide reliable analysis.

5. Protein Attachment (Direct) Non-covalent: hydrophobic, van der Waals, hydrogen bonding, electrostatic forces, etc Advantage: no chemical modification is required Disadvantage: may get denatured (Indirect) Covalent: bifunctional crosslinker Thiol (-SH3) on gold substrate Silane on glass and silicon substrate (SiO2) Non-covalent: advantage is their ease of application since no chemical modification is required. Chemisorbed with nearly crystalline packing Non-covalent: advantage is their ease of application since no chemical modification is required. Chemisorbed with nearly crystalline packing

6. Outline Techniques --Tommy Photolithography Soft lithography Microcontact printing Microfluidic network Applications -- Matt Cell growth Cell migration 3D Mcirofluidic network

7. Photolithography Chrome mask can be 20-100 times more expensive than transparency Chrome mask can be 20-100 times more expensive than transparency

8. Photolithography Dominant technique for patterning solid state devices Requires clean room facilities Chemicals used can denature biomolecule Draw backs: Intrinsically expensive Limited control over surface protpertites Often not directly applicable to proteins and cells Unfamiliar and inaccessible to most biologists. Draw backs: Intrinsically expensive Limited control over surface protpertites Often not directly applicable to proteins and cells Unfamiliar and inaccessible to most biologists.

9. Soft Lithography Whitesides and colleagues ’98 A soft elastic stamp, Polydimethylsiloxane (PDMS) Common Techniques: Microcontact printing (µCP) Microfluidic patterning Stencil patterning Whitesides and collegagues developed a set of techniquess which is more biocompatible for patterning biomoleculres. Hexadecanethiol on gold Octadecyltrichlorosilane on SiO2 octadecanethiol (ODT) Polydimethylsiloxane (PDMS) Si-O-Si-O-Whitesides and collegagues developed a set of techniquess which is more biocompatible for patterning biomoleculres. Hexadecanethiol on gold Octadecyltrichlorosilane on SiO2 octadecanethiol (ODT) Polydimethylsiloxane (PDMS) Si-O-Si-O-

10. MicroContact Printing Replica molding Transfer by conformal contact Simple and inexpensive method Compatible with many molecules Nonplanar surfaces Multilayers Alkanethilos on film of gold and silver. Alkylsiloxanes on hydroxylated surfaces of glass and silicon dioxide. Multiple monolayer by repeated application Monolay 30, max van der waals interaction Alkanethilos on film of gold and silver. Alkylsiloxanes on hydroxylated surfaces of glass and silicon dioxide. Multiple monolayer by repeated application Monolay 30, max van der waals interaction

11. Micro Fluidic Network Characteristics: 10-100 micron, 0.1cm/s, milisecond for diffusion and reaction times Capillary force Pressure assisted flow Suitable for patterning delicate materials (protein and cells) Capillary forces driven flow is limited to small areas and channels and it is not suitable for viscous fluid. Y micro fluidic device -decreased cost in manufacture, use and disposal -reduced consumption of reagents and analytes -increased portability -similar to biological condition? more accurate info Drawbacks: susceptible to blockages from particles, more sensitive to adsorption of species of the surfaces. Largely laminar flow, basis for useful fluidic decices Capillary forces driven flow is limited to small areas and channels and it is not suitable for viscous fluid. Y micro fluidic device -decreased cost in manufacture, use and disposal -reduced consumption of reagents and analytes -increased portability -similar to biological condition? more accurate info Drawbacks: susceptible to blockages from particles, more sensitive to adsorption of species of the surfaces. Largely laminar flow, basis for useful fluidic decices

12. Drawbacks Lost normal functionality upon attachment to substrate Limitation of the number of proteins can be patterned

13. Outline Techniques --Tommy Photolithography Soft lithography Microcontact printing Microfluidic network Applications -- Matt Cell growth Cell migration Other Applications

14. Cell Growth and Viability Binding to the extracellular matrix (ECM) controls local differentiation in capillaries Disruption in ECM leads to cell death Allowing suspended cells to bind antibodies to integrins prevents apoptosis However in vivo studies have shown that dying capillary cells remain in contact with the ECM Cells grow and spread on large beads (>100mm) and die on smaller beads (4.5mm)

15. However small beads can be engulfed into the cells complicated the analysis Micropatterned square fibronectin adhesion islands onto gold Vary the island size and measure growth and apoptosis Cell Growth and Viability Cont.

16. Geometric Control of Cell Life and Death Increase the area of patterned square from 75 to 300 um2 Apoptosis declines DNA synthesis increases However this maybe due to more integrin binding, focal adhesion formation, or greater accessibility to growth factors in the matrix

17. Modify experiment Arrange closely spaced islands of 3 to 5 um in diameter. Keep the area of contact the same but vary the spacing As projected area increases DNA synthesis increases Apoptosis decreases

18. Experimental Conditions Cell type Human capillary endothelial cells Proteins patterned Fibronectin Antibodies to integrin b1 and integrin avb3 Collagen (binds b1) Vitronectin (binds avb3) Observation Contacts specific to b1 lead to apoptosis more than contacts to avb3

19. Conclusion Findings Cell size can control whether a cell grows or dies regardless of ligand However adhesion of different receptors determines how sensitive the cell is to size Possible Explanations Focal adhesions may orient the signal transduction machinery of the cell Growth and survival might be directly linked to the mechanical stress

20. Cell Migration It is known that chemoattractants and integrin receptors can contribute to cell migration Do the mechanical forces within a cell alter the mechanism of cell migration?

21. Experiment Micropattern different ECM components in different shapes with microcontact printing Fibronectin, collagen, thrombospondin-1 squares and circles Treat cells with growth factors PDFG and FGF Observe how the cells extend lamellipodia

22. Finding Circle random extension Square extension from the corners Occurs for endothelial cells on fibronectin and myoblasts on thrombospondin-1 each patterned on glass Growth factor receptors are still homogenously distributed

23. More observations Stress fibers on square islands orient themselves with the diagonal of the cell Vinculin concentrated at corners indicated focal adhesions are at the corners Do physical constraints cause cells to focus traction forces at the corners?

24. Traction Force Microscopy Used to measure tension in the cell Collagen islands on polyacrylamide gel with imbedded 2mm fluorescent beads Culture human airway smooth muscle cells Greatest displacement was at the corners

25. Possible Explanation Local tension transfer locally activate Rho GTPases or CDC42 Known previously Rac can be active globally but needs effectors that colocalize with integrins Cell generates mechanical forces which causes the arrangement of stress fibers and focal adhesions which allow for the local activation of lamellipodia extension by Rho Rac and CDC42 Mechanical forces are important for migration direction

26. Other Application Use microfluidics to create stable and easily producible concentration gradients Cell-cell interactions in cocultures Cell shape can control stem cell differentiation High through put assays Optimize culture conditions Yield information about cell fate decisions Microfluidics and layer approaches to develop 3D environments for tissue engineering

27. “Thus, the continued merger of engineering, medicine, materials, and biological sciences as mediated by microscale approaches in tissue engineering and biology will enhance our ability to create in vivo like physiological models that can be used for fabricating tissues or for understanding fundamental biology”

28. Conclusion and Outlook Softlithography can be carried out conveniently, rapidly, and inexpensively. Pattern delicate biological matter and is applicable to wide range of materials Can be applied to many biological aspects such as Immunoassay Tissue Engineering Biosensors Incorporation of micro or nanoparticles assembly creasts a nonplanar topography for protein and cell attachment. Non-planar increases the density of biomolecules. Similar to the size of the dimention of protein, the protien may adsorbed to the surface with less interaction and will be able tot better retain its structure and bioactivity. Incorporation of micro or nanoparticles assembly creasts a nonplanar topography for protein and cell attachment. Non-planar increases the density of biomolecules. Similar to the size of the dimention of protein, the protien may adsorbed to the surface with less interaction and will be able tot better retain its structure and bioactivity.

29. References Delamarche et al, Advanced Materials. 2005 Whitesides et al, Annu. Rev. Mater. Sci 1998 Yap et al, Biosensors and Bioelectronics. 2007 Bernard et al, Advanced Materials, 2000 Mrksich et al, Annu. Rev. Biophys. Biomol. Struct. 1996 Takayama et al. 1999. Proc. Natl. Acad. Sci. USA

  • Login