Cell Manipulation on a Well Defined Phosphorylcholine Polymer Brush

Adsorption of fluorescein-isothiocyanate-labeled BSA was well controlled on a patterned graft polymer surface, as shown in Fig. 15. On an UV-irradiated region with no polymer brush, the fluorescence intensity was significantly high, indicating that a large amount of BSA was adsorbed in this region. Conversely, BSA adsorption was remarkably reduced in the

Mesh size of photo-mask

50 um

Polymerization time: 18 h Polymer thickness 12 nm

Fig. 15. Fluorescence micrographs of fluorescein isothiocyanate-labeled albumin adsorption on patterned poly(MPC) brush surface after contact with 0.45 g/dl FITC-albumin in PBS for 30 min. Reprinted with permission from Iwata et al. (2004), copyright (2004) American Chemical Society poly(MPC) brush layer. Figure 16 shows the fibronectin adsorption pattern on thepattern surfaceafter contactwiththecell culturemediumfor 60 min. The adsorption pattern was determined by immunoassay. On the polymer brush prepared by polymerization for 10 min, fluorescence caused by the adsorbed fibronectin was observed to be homogeneous. The difference in fibronectin adsorption on the patterned surface was clearly related to the

Polymerization time

Fig. 16. Fluorescence micrographs of fibronectin adsorption on a patterned poly(MPC) brush surface after contact with cell culture medium for 30 min. Reprinted with permission from Iwata et al. (2004), copyright (2004) American Chemical Society. L Large, M medium, S small

Mesh size of photo-mask: S 50 um

Fig. 16. Fluorescence micrographs of fibronectin adsorption on a patterned poly(MPC) brush surface after contact with cell culture medium for 30 min. Reprinted with permission from Iwata et al. (2004), copyright (2004) American Chemical Society. L Large, M medium, S small time of polymerization of the polymer brushes. On the poly(MPC) brush surface, fibronectin adsorption was effectively reduced. The reduction of plasma protein adsorption on MPC polymer surfaces has been reported previously, and the mechanism is considered to be related to the structure of the polymer surface (Ishihara et al. 1998).

The study to clarify the difference in surface structure between a PMB coating and the dense poly(MPC) brush prepared in this study on the reduction of protein adsorption is still ongoing. However, it has been shown that poly(MPC) brushes just 5 nm thick prepared by the "grafting from" system can reduce protein adsorption. Although the thickness of the cast film of PMB that we made has been normally controlled on a submicron scale, the poly(MPC)-brush thickness can be controlled on the scale of a few nanometers. This is a great advantage for surface modification to improve the nonfouling properties of micro- or nanodevices.

Figure 17 presents fluorescence micrographs of fibroblasts that adhered onto the patterned surface. The effect of surface grafting of poly(MPC) by polymerization for 10min on fibroblast adhesion was not observed and the cells adhered homogeneously to the surface. By adjusting the time of polymerization, cell adhesion was controlled and fibroblasts adhered to UV-irradiated regions that had no poly(MPC) brushes. This result is coincident with protein adsorption on the surface. Above a poly(MPC)-brush thickness of about 5 nm, protein adsorption and cell adhesion was remarkably reduced (i. e., they were able to recognize the thickness of the thin brush). The effect of the grafting density of poly(ethylene glycol) on protein adsorption has been reported by Sofia and coworker (1998). Grafting density may also be important for controlling protein adsorption on poly(MPC)-brush surfaces.

The number of adherent cells can also be controlled with a change in the surface area of the UV-irradiated region. The surface area of the pattern affected cell density. The surface areas for adherent cells on small-and medium-sized pattern surfaces were 844 ± 185^m2/cell and 1188 ± 240 ^m2/cell, respectively. The large-sized pattern surface had a cell density of 159 ± 34 ^m2/cell, which is significantly higher. Cell communication depends on the area of the surface where the cells are able to adhere.

To fabricate a microscale pattern on a solid surface, microcontact printing and soft lithography techniques have been applied. Whitesides and coworkers reported that microscale patterning provides a versatile method for creating novel adhesive substrates that are useful for spatially positioning mammalian cells and controlling their viability, form, and function (Brock et al. 2003; Chen et al. 1997; Singhvi et al. 1994).

Armes and coworkers have reported that the molecular architectures of MPC polymers can be easily controlled with ATRP in protic solvents (Li et al. 2003; Lobb et al. 2001; Ma et al. 2002), with the result that a wide vari-

Polymerization time

Polymerization time

Fig. 17. Fluorescence micrographs of fibroblast adhesion on patterned poly(MPC) brush surface after incubation for 24 h. Concentration of fibroblasts, [Fibroblast] = 5.0x 104 cells/ml. Reprinted with permission from Iwata et al. (2004), copyright (2004) American Chemical Society

ety of polymer surface designs will be possible. Surface modification with well-defined MPC polymers would be considered as one of the most robust methods with which to optimize biointerfaces on a molecular scale. Microfabrication with MPC polymers might prove to be important in separations, biosensors, and the development of biomedical materials.

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