| Summary: | 碩士 === 國立清華大學 === 工程與系統科學系 === 102 === The interactions between cells and cells play essential roles in cell biology. Nowadays reports showed that studying cells to cells interactions is an important research to understand cell behaviors such as variation or tumor progression and metastasis, which cannot be fully revealed by traditional methods based on the ensemble average behaviors of large population of cells incubated in petri dish. Thus single cell positioning, incubation and observation have become an important technique to analyze single cell behaviors. In this study, we proposed an unique chip combining micro snare to capture single cells and controllable valves to manipulate fluid operation, for facilitating the study of single cell-cell interactions.
In this research, a process consisting mechanical traps and valves was presented for studying single-cell interactions. Firstly, two single-cells were captured independently by the 15μm x 15μm snare which was designed as the diameter of the cell. Afterward, the two single-cells underwent perfusion culture. After both cells adhered to the bottom of the channel, cell A was stimulated by some chemicals and released some substance. Then the substance released by cell A stimulates cell B by opening the path between them. Finally, the substance released by cell B passed into the detection part to examine the variation of cell B.
We used simple technique to fabricate our chip, including microfluidic channel, controllable valve and cell trap. Moreover, the valves could be manipulated on-off by a vacuum pump controlled via Lab-view software (National Instruments). The testing of the four series valves exhibited the precise operation and control of the valves. Certain fluid was
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injected into two different directions to ensure the valves could switch the pathway between two single-cell culturing systems. In this way, the two single-cells could be cultured under independent medium route avoiding mutual mixture and could also be affected by each other by opening the valve between two systems.
After single cell trapped inside the snare, the streamline will not go through the snare center since the increase of the flow resistance and the rest of cells will pass by without accumulation inside the trap. Hela cells dyed with Calcein AM Fluorescent were loaded into the chip and the single-cell was captured by the trap. Discovering captured cells simultaneous in our unique chip is feasible. Finally, we simulated thermosensitive liposome as one of single-cell (cell A), and induced liposome releasing some fluorescent dye to affect another real single-cell (cell B). The fluorescent change showed that the second single-cell apparently received the signal released by liposome from the cell A channel. Thus, the way as the third testing, Doxorubicin was encapsulated by liposome. Heating up the temperature made the liposome release Doxorubicin to stimulate another single-cell. Finally, we examined the stimulated single cell viability by Calcein. The above four serial experiments not only prove the completeness of the chip but reveal its biological feasibility. We could further use this special design to undergo cell-to-cell mutual interactions in the future. It is a prospective platform for cell-cell interaction research, which can also combine with biological or electrochemical detection someday.
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