1. MICROFLUIDIC SPATIAL CONTROL OF STEM CELL DIFFERENTIATION J. Kawada 1,3, H. Kimura 1,3, H. Akutsu 2,3, Y. Sakai 1,3 and T. Fujii 1,3 1 Institute of Industrial Science, The University of Tokyo, Tokyo, Japan 2 National Research Institute for Child Health and Development, Tokyo, Japan 3 JST CREST, Tokyo, Japan 3 JST CREST, Tokyo, Japan 14th International Conference on Miniaturized Systems for Chemistry and Life Sciences 3 - 7 October 2010, Groningen, The Netherlands 授課老師 : 劉承賢 學生 : 陳冠宇

2.  Abstract  Introduction  Material and Method  Experimental  Results and Discussion  Conclusion Outline

3.  Develop: A technique to control differentiation of mouse pluripotent stem cells by generating chemical distributions in the culture environment on a microfluidic device.   Chemicals injected into the lower channel are transported through the membrane to the upper channel.  Demonstrated: The differentiation of mouse induced Pluripotent Stem Cells (iPSCs) can be spatially controlled according to the laminar flow patterns in the lower channel. Abstract

4.  Pluripotent stem cells (PSCs): Have the potentials to indefinitely proliferate and to differentiate into derivatives from three germ layers   For the purpose, it is required to establish the way to intentionally induce differentiation into specific cells.  There is almost no ways to control the spatial distribution of molecules in the conventional culture vessels. Introduction

5.  Microfluidic method have been proposed for exposing cultured cells to chemicals in spatially resolved ways.  The present study aims at the development of a microfluidic method to generate stable chemical distribution over a long period of time. Introduction

6. Material and Method  The key component: --Lower channel for controlling fluid --Upper channel for cell culture

7. Material and Method 

8.  The distribution of the chemical concentration generated in the upper channel was evaluated by measuring the fluorescence intensity of FITC and GFP.  Result of fluorescence intensity along the channel width:

9. Experimental  Two different patterns:

10. Experimental  First, the cells suspension (107 cells / mL) was injected into upper channel  Then its inlet and outlet were closed.  After the seeded cells attached to the membrane coated with fibronectin, two types of media --Leukemia inhibitory factor (LIF) --Retinoic acid (RA) were injected into the lower channel at 2 µL/min each.   The miPSCs seeded in the channel are exposed to these factors and exhibit their response according to their positions in the channel.

11. Results and Discussion  On the day 4 of culture, GFP and DAPI fluorescence intensity in the upper channel were measured  --As compared with fluoresceince intensity distribution of GFP on the day 1 --miPSCs could be differentiated locally following the laminar flow patterns formed in the lower channel --while the distribution of the cells stayed spatially constant

12. Results and Discussion

13.  In Figure 5(b), the fluorescence intensity is decreasing along the channel width meaning that the cells located in the left side of the channel are differentiated and the expression of Nanog is lowered.   In Figure 5(c), the fluorescence intensity in the middle of the channel is higher than the area close to the side walls   By changing the laminar flow pattern in the lower channel, differentiation states of miPSCs could be controlled in a position- dependent manner. Results and Discussion

14.  Developed: A method to control the differentiation of PSCs, using a device having two compartmentalized channels (lower and upper) separated by the porous membrane.  Demonstrated by using the method: Position-dependent differentiation patterns of miPSCs were successfully controlled according to the laminar flow patterns formed in the lower channel.  Advantage of the device: To generate the static chemical distribution by using compartmental channels and the porous membrane. Conclusion

15. Thank you