| Summary: | 碩士 === 臺灣大學 === 應用力學研究所 === 95 === Multiple single human bone marrow stem cells with dynamic control of perfusion have been demonstrated in an array of individually isolated microenvironments of a microfluidic device and its associated system. In addition to micro device fabrication, the present study investigates living cells, and monitors the real-time living cell processes in a micro device such as expansion and differentiation of single or several human bone marrow stem cells in individual microenvironments, thus providing a quantitative description of live stem cellular behavior. Such a work, however, has been hampered by the difficulty in data collection using traditional techniques. There is much interest in quantifying the range of biological responses of individual cells to various physiologically-relevant stimuli as opposed to bulk averages. Particularly useful information can be acquired if the environmental factors that contribute to variable responses for individual cells are controlled. Bulk experiments fall short of providing adequate data in that the results elucidate the mean value of a parameter with further experiments providing information about the standard error of the mean. In contrast, single-cell experiments reveal the critical underlying distribution of parameters. This contrast is especially apparent in time-dependent processes such as expansion and differentiation of stem cells in which by averaging over a population. A smooth transition is observed from one state to another, which used to obscure the underlying transition occurring at the cell level. Perfusion culture has been previously used and been able to reduce the effects of diffusible elements on cell behavior by convecting away produced substances that may provide autocrine or paracrine signals. Microfluidics approaches have been developed to allow more precise control of cell positioning and reagent introduction in analyzing single or rare cells.
KP-hMSCs (stem cell line derived from human bone marrow) were used in the experiments. Freshly suspended cells were introduced into previously PBS filled microfluidic devices by a syringe connected to a three way valve. Single KP-hMSC was trapped and maintained in individual micro-well by using microfluidic perfusion for dynamic cell culture and differentiation induction. Cells were both cultured in an incubator between images, and were also maintained at 37℃ on a microscope stage for time-lapse experiments.
We demonstrate the culture of single KP-hMSC under constant perfusion of media + 20% FBS. In a flow rate of 5μL/min time-lapsed images were taken every minutes of an isolated micro-well of single KP-hMSC on an incubated microscope stage. After one hour, slight changes in morphology were observed away from a spherical morphology towards an adherent morphology. Also, cell division was found in a few cases. After 12 hours, a majority of single cells displayed adherent morphology, and crawled out of the microenviroment by extending the filopodia.
We have demonstrated that quantitative analysis of the dynamics of cell adhesion, death, division, and escape from traps were performed in the time length of 72 hours. It was also found that 60% of cells displayed adherent morphology after 24 hours. In 48 hours, 20% of cells showed characteristics of apoptosis, while 10% had escaped from the vicinity of the initial trapping site and 10% of cells had undergone cell division.
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