Although single cell manipulation has been developed for precise understanding of cell biology, it is still difficult to handle single cells because of their morphology including the size variation and the flexibility. We developed a single cell manipulation device featuring microchannels and micropockets for single cell capture and cultivation. Single cells were captured noninvasively and sequentially, and each cell was repeatedly sub-cultured to four generations. The single cell manipulation devicewas microfabricated with two main parallel channels allowing the cell suspension and the carrier flow to be injected separately. Those channels, that are main channel and buffer channel, were connected with a narrow (3μm) drain channel, and single cell capture pockets were placed at the point where the main channels and the drain channel connected. A gentle flow was produced in the drain channel because of the difference in the flow rate between the main channel and buffer channel, realized the individual single cell isolation in the capture pocket. When a single cell was captured in a single cell pocket, the captured cell capped the drain channel and caused the other cells that were flowing through the channel to go to the next capture pockets. The ratio between the cells thatwere captured and the cells that passed through the main channel was about 70%. The captured singe cellwas cultured successively in the same pocket, and the cells divided into four cells. The doubling times of two cells that grew in the first cell division were slightly different (10 min). These fundamental single cell manipulations were carried out mainly by controlling the flow rate, essentially the pressure on each channel. Occasionally the manipulations also were carried out by changing the shapes and the sizes of the micropockets in the microfluidic device. Since this device was used successfully for the noninvasive isolation and long-term cultivation of single cells, it can contribute to various biological analyses, such as biopsy, noninvasive bioanalysis, and the morphology of single cells.
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