(459e) Parallel Manipulation of Adhering Living Cells Based on Photo-Induced Cell Capturing

In order to meet the diversifying demand for the cell manipulation in the rapid progress of cell engineering, we proposed a concept of photo-manipulation of living cells in adhering state as a principle of novel biotool, and have been promoting the development of elemental technologies to implement the concept. In clear contrast to the conventional cell patterning using a previously patterned substrate, the area to retain the cells can be defined after cell seeding, and the captured cells can continue to grow freely beyond the defined area in further incubation. Up to now, we have developed several substrates having photoresponsive cell adhesion, and reached the finding of the conditions to capture living cells firmly onto the substrate containing no photoresponsive compound just by irradiating light.

In this study, we demonstrate cell pattern formation and cell arrangement in a closed microchannel by using a newly developed apparatus, which enables us to project arbitrary micropattern onto the surface of culture substrate under microscopic observation. Further, we investigate the characteristics of photo-induced cell capturing in order to examine the feasibility of parallel manipulation of living cells based on this novel technology.

In order to compose a cell manipulation system based on photo-induce cell capturing, we developed a PC-controllable microprojection unit and installed it in the optical system of an inverted microscope. We determined that the system can project the light with the wavelength of 365 nm, which induced cell capturing and cannot be irradiated with the existing microprojection system based on commercialized LCD projector, in arbitrary pattern with the resolution of 7 micron in the area of 3.7 mm x 2.7 mm.

For the experiment of cell pattern formation, we used CHO-K1 cells, which had been seeded uniformly on a fibronectin-coated 35mm-diameter culture dish , and cultured for 24 h. We irradiated the light with the wavelength of 365 nm at focusing a computer-generated micropattern at the bottom surface of the cell culture dish. Then the culture medium was substituted by phosphate buffer saline (PBS) containing 1 mM EDTA. After 10 min, we flushed the bottom surface of the dish with PBS to remove the weakly adhering cells.

Figure shows the microscopic images of the CHO-K1 cells, which were micropatterned by using a newly developed cell manipulation system based on the photo-induce cell capturing. We obtained the highly contrasted cell patterns indicating that the cells were captured firmly on the cell culture dish in the irradiated region, while the cells, of which the adhesion was weakened through the EDTA treatment, were removed completely from the non-irradiated region. Using the cell maniplation system, also we succeeded to arrange two types of cells in prescribed positions in a closed microchannel.

In the examination of the characteristics of photo-induced cell capturing, it was clarified that the photo-induced cell capturing is basically temporal and can be reset by leaving for 10 hours. The reversibility of the photo-induced cell capturing was considered to provide the cell manipulation based on this principle with valuable flexibility. Further, it was also confirmed that the cells, which had been captured and retained in a confined area by local light irradiation, continued to grow vigorously beyond the light irradiated region through subsequent incubation. In addition to the freedom to select the cell retaining area after cell seeding, this feature of the photo-induced cell capturing is expected to contribute to the feasible process to purify and multiply the cells expressing some specific property or successfully transduced cells , for instance.

From these experimental results and the intrinsic compatibility with electronics and information technology, it was suggested strongly that the parallel cell manipulation based on the photo-induced cell capturing is feasible to provide the research field of cell engineering with a useful and powerful tool.

Acknowledgement This work was supported by the Industrial Technology Research Grant Program in 2005 from the New Energy Development Organization (NEDO) of Japan, and Creation and Support Program for Start-ups from Universities in 2005 from Japan Science and Technology Agency.

References Edahiro, J.-I., Sumaru, K., Tada, T., Ohi, K., Takagi, T., Kameda, M., Shinbo, T., Kanamori, T., Yoshimi, Y., 2005. In-situ control of cell adhesion using photoresponsive culture surface Biomacromolecules, 6, 970?974. Tada, Y., Sumaru, K., Kameda, M., Ohi, K., Takagi, T., Kanamori, T., Yoshimi, Y., 2006. Development of a photoresponsive cell culture surface: regional enhancement of living cell adhesion induced by local light irradiation. J. Appl. Polym. Sci. 100, 495?499.