Cell-Environment Interactions. Chieh-Chun Chen

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1 Cell-Environment Interactions Chieh-Chun Chen

2 Part 1: Soft Lithography in Biology and Biochemistry Chieh-Chun Chen

3 Outlines Introduction Key features of soft lithography Applications In microscopic biochemical assays In cell biology

4 Introduction Photolithography Patterning of species on surfaces with micro scale resolution to fabricate integrated circuits. Limitations Expensive Limited control over surface properties Not directly applicable to proteins and cells Time from a design to prototype is long unfamiliar to most biologists

5 Introduction Soft lithography Uses elastomeric materials to fabricate the pattern by molding Enables to have complex biochemical patterns Advantage Convenient, rapid and inexpensive control the properties of surfaces at the molecular level

6 Introduction Potential Applications Cellular studies Tissue engineering Development of biosensors Immunoassay Delamarche et al, Advanced Materials. 2005

7 Key Features of Soft lithography Surface Engineering Self-assembled monolayers (SAM) Alkanethiolates HS(CH 2 ) n X on gold The properties of the surface mostly determined by X group Inert surfaces Resist the adsorption and adhesions of biological species Ex. Cover the surface with Oligo (ethylene glycol)

8 Key Features of Soft lithography Surface Engineering Attaching ligands Using a surface with low density of ligands and otherwise inert forms mixed SAMs Increase specificity Without interference from other ligands Without nonspecific interactions with other substract

9 Key Features of Soft lithography Surface Engineering Attaching ligands Homogeneous SAM v.s. hetergeneours SAMs

many molecules Multilayers Whitesides, Annu. Rev. Mater.

10 Key Features of Soft lithography Replica molding Transfer by conformal contact Simple and inexpensive method Compatible with many molecules Multilayers Whitesides, Annu. Rev. Mater. Sci 1998 Yap, Biosensors and Bioelectronics Bernard, Advanced Materials, 2000

11 Key Features of Soft lithography Poly(dimethylisiloxane) Stamps (PDMS) for replica molding Stiff elastomer Nontoxic and readily available commercially

12 Key Features of Soft lithography Microcontact Printing (μcp) Inking the stamp with a solution; alkanethiolate ink is deposited contact with the surface

13 Key Features of Soft lithography Elastomeric Membranes for Dry Lift-off

14 Key Features of Soft lithography Microfluidic systems do not mix turbulently but flow in adjacent mix only at their interface by diffusion

15 Key Features of Soft lithography Microfluidic systems Silver wire as a reference electrode by Precipitation

16 Key Features of Soft lithography Microfluidic systems Patterning Using 2D Microfluidic Structures Channel systems Patterning Using 3D Microfluidic Structures generate complex patterns of different materials by deposition from solution.

17 Key Features of Soft lithography Microfluidic systems Generation of Gradients Allowed to mix diffusively

18 Applications in Biochemical assays To miniaturize biochemical assays Minimize the amounts of analytes Maximize of the number of assays in parallel Microfluidic structures Pumps, Valves, Mixers, Detectors

19 Applications in Cell Biology Control the spatial arrangement of surface Isolation of cells => study each individual cell Prevent cells from migration => enable to study single cell repeatedly Not complicated by cell-cell interactions Disadvantage

20 Applications in Cell Biology Patterned Cell Culture

21 Applications in Cell Biology Pattern cells without SAMs, Study of cell spreading

22 Applications in Cell Biology Cells attached in arrays of microwells

23 Applications in Cell Biology Coculture Hepatocytes and fibroblasts Patterning over a single cell The medium Inside a cell Cell substrate

24 Summary Soft lithography Surface engineering: SAMs, inert surface, ligand binding PDMS, Microcontact Printing Applications Microfluidic systems Patterns in Cell culture, Coculture, over a single Cell

25 Part 2: Geometric Control of Cell Life and Death

26 Outlines Observations Question Experiments

27 Cell Growth and Viability Observations 1. Binding to the extracellular matrix (ECM) controls local differentiation in capillaries 2. Disruption in ECM leads to cell death 3. Allowing suspended cells to bind antibodies to integrins prevents apoptosis Adhesion-dependent control of apoptosis has been assumed to be mediated by changes in integrin signaling.

28 Cell Growth and Viability Observations 1. Dying capillary cells remain in contact with the ECM 2. Cells grow and spread on large beads (>100mm) and die on smaller beads (4.5mm) 3. However small beads can be engulfed into the cells complicated the analysis Cell shape might related to cell growth and death.

29 Question Whether cell shape or integrin binding per se governs life and death in these cells?

30 Experiments Micropatterned square fibronectin adhesion islands onto gold Vary the island size and measure growth and apoptosis

31 Experiments Cells became more rounded as bead diameter decreases 60% suspended cells die

32 Experiments The size of beads affect not only the degree of cell but also ECM curvature and bead internalization

33 To determine the precise spreading requirements for survival rather than growth, using different-sized islands to test Experiments

34 Experiments Increase the area of patterned square from 75 to 300 um 2 Apoptosis declines DNA synthesis increases However this may be due to more integrin binding, focal adhesion formation, or greater accessibility to growth factors in the matrix

35 Experiments Arrange closely spaced islands of 3 to 5 um in diameter. Keep the area of contact the same but vary the spacing (Fix Cell-ECM areas)

36 As projected area increases DNA synthesis increases Apoptosis decreases cell shape per se appears to be the critical determinant Experiments Chen, CS et al. Science v276 p1425

37 Experiments Results Apoptosis was greatly inhibited relative to the 60% level observed in suspended cells Contacts specific to β 1 lead to apoptosis more than contacts to α v β 3 Fibronectin Antibodies to integrin b1 a integrin avb3 Collagen (binds b1) Vitronectin (binds avb3)

38 Results Conclusion 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

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